3-aryl propiolonitrile compounds for thiol labeling

ABSTRACT

The present invention relates to a process for labeling compounds comprising thiol moieties with 3-arylpropiolonitrile compounds, to 3-arylpropiolonitrile compounds substituted with tag moieties and to specific 3-arylpropiolonitrile linkers.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the National Stage of International Application No.PCT/EP2014/064387, filed on Jul. 4, 2014, which claims the benefit ofEuropean Application No. 13305950.1, filed on Jul. 4, 2013. The contentsof both applications are hereby incorporated by reference in theirentirety.

The present invention relates to a process for labeling compoundscomprising thiol moieties with 3-arylpropiolonitrile compounds, to3-arylpropiolonitrile compounds substituted with tag moieties and tospecific 3-arylpropiolonitrile linkers.

BACKGROUND OF THE INVENTION

Over 90% of the human proteins contain cysteines, while in silico digestof the human proteome revealed that only about 15% of all human trypticpeptides detectable by mass spectroscopy (MS) contain at least onecysteine in their sequence. This observation combined with the presenceof a highly reactive thiol group on its side chain makes cysteine anattractive target for chemical labeling. Cysteine is the only codedamino acid that carries a nucleophilic sulfhydryl (or thiol) group(—SH), which largely exceeds the reactivity of any other nucleophilicfunction susceptible to be present in proteins. As a result,chemospecific cysteine derivatization is by far the most widely usedmethod for chemical tagging of proteins. Among the vast number ofchemical cysteine modification methods reported in literature so far,reagents such as N-substituted maleimides, 4-vinylpyridines andiodoacetamides are most commonly used. All of them possess drawbackspreventing them from being ideal methodology for cysteine labeling,though being suited for this task. These drawbacks are mainly presenceof undesired side reactions, in particular for iodoacetamides andmaleimides, and instability of addition product in biologicalenvironments due to reversible thiol exchange and other side reactions.

The present invention relates to a process for labeling compoundscomprising at least one thiol moiety, such as cysteine, with compoundscomprising a tag moiety and a 3-arylpropiolonitrile moiety. Saidcompounds and their addition products with cysteine derivatives show anunexpected stability in a wide range of conditions. The process forlabeling compounds comprising thiol moieties of the invention can thusbe used for a wide range of applications.

SUMMARY OF THE INVENTION

The first object of the invention is a process for the preparation of alabelled compound comprising a thiol moiety, comprising contacting acompound comprising a thiol moiety with a compound of formula (I)

wherein R₁ to R₅ are as described below, and wherein at least one of R₁to R₅ comprises a tag moiety.

Another object of the invention is a compound of formula (I)

wherein R₁ to R₅ are as defined below, and wherein at least one of R₁ toR₅ comprises a tag moiety.

Another object of the invention is a compound of formula (II)

wherein R₁ to R₅ are as defined below, and wherein at least one of R₁ toR₅ is different from a hydrogen atom.

Another object of the invention is a compound of formula (III)

wherein R₆—S corresponds to the moiety of the compound comprising atleast one thiol moiety as identified above.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Evolution of intensity of fluorescence over time obtained inhuman plasma for the same concentration of compounds A and B.

FIG. 2a-2c : a) Evolution of intensity of fluorescence over timeobtained in cellulo for the same concentration of compounds A and B.

-   -   b) Intensity ratio compound B/compound A at different times in        cellulo.    -   c) Microscope pictures of cells treated with compounds A and B,        superimposition of TAMRA and Hoechst labeling. White spots        around nucleus correspond to hydrolyzed probe.        A—arylpropiolonitrile probe, B—maleimide probe.

FIG. 3a-3b : HPLC monitoring of the hydrolysis of phenylmaleimide (2) inPBS (1×, pH 7.6) at 1 mM concentration at 25° C.; the peaks correspond,from the earliest to the latest, to

conversion was monitored by disappearance of the starting material (a).Pseudo first order rate constant for the reaction was determined byplotting the ln([phenylmaleimide]) versus time and analyzing by linearregression. The constant corresponds to the absolute value of determinedslope (b).

FIG. 4: HPLC monitoring of hydrolytic stability ofN-(4-(cyanoethynyl)phenyl)acetamide (pNHAc-APN, 11) in PBS (1×, pH 7.6)at 1 mM concentration at 25° C. Conversion was monitored bydisappearance of the starting material; the peaks correspond, from theearliest to the latest, to

No detectable conversion of arylpropiolonitrile 11 was observed after 5hours of monitoring.

FIG. 5: MTT test results for compounds 1, 2, 3, 5, 7, 11, 10 and 9.

FIG. 6a-6c : a) schematic structure and measured DLS spectra of CD38 andCD38 A275C mutant; b) scheme of CD38 A275C modification with 49; c) DLS(Dynamic Light Scattering) spectrum of the resulting conjugate.

FIG. 7a-7b : Gel electrophoresis of CD38-C375 mutant labeled with acompound according to the invention and with the corresponding maleimidecompound, before purification (a) and after purification (b).

FIG. 8: Strategy of preparation of the antibody-TAMRA conjugate with acompound according to the invention, and the comparison with thecorresponding maleimide.

FIG. 9: Gel electrophoresis of the conjugates obtained respectively withthe compound according to the invention (CBTF) and with thecorresponding maleimide (SMCC).

FIG. 10: Mass spectrum for the conjugate obtained with the compoundaccording to the invention.

FIG. 11: Zoom on the mass spectrum of FIG. 10.

FIG. 12: Mass spectrum obtained with the maleimide.

FIG. 13: Zoom on the mass spectrum of FIG. 12.

FIG. 14: General scheme of direct conjugation of the compound 58 topartially reduced Trastuzumab.

FIG. 15: SDS-PAGE analysis of the obtained conjugates shows thatcompound 58 is covalently attached to the antibody

FIG. 16: General scheme of rebridging of antibody fragments usingcompounds 33 and 34.

FIG. 17: SDS-PAGE analysis shows that antibody fragments aresuccessfully bridged by compounds 33 and 34.

DETAILED DESCRIPTION OF THE INVENTION

The first object of the invention is a process for the labeling of acompound comprising a thiol moiety, comprising contacting said compoundcomprising a thiol moiety with a compound of formula (I)

wherein each of R₁ to R₅ is independently selected in the groupconsisting of:

-   -   hydrogen atoms,    -   alkyl, alkene or alkyne groups, optionally interrupted by at        least one heteroatom selected among O, N and S,    -   aryl groups,    -   alkoxy groups,    -   halogen atoms,    -   amino (—NRR′) groups, wherein R and R′ are independently        hydrogen atoms, alkyl, alkene, alkyne or aryl groups as defined        below,    -   hydroxylamine (—ONH₂) group,    -   hydrazine (—NH—NH₂) group,    -   nitro (—NO₂) group,    -   azido (—N₃) group,    -   diazonium (—N₂ ⁺) group, optionally in presence of a counterion,    -   maleimide group,    -   alkyl- or aryl-carboxyl (—C(═O)OR) groups, wherein R is as        described above,    -   alkyl- or aryl-carbonyl (—C(═O)R) groups, wherein R is as        described above,    -   hydroxyl (—OH) group,    -   boronic acid —B(OR″)₂ group, wherein R″ is a hydrogen atom or an        alkyl group,    -   phosphine or phosphonium groups,    -   isocyanate (—N═C═O) or isothiocyanate (—N═C═S) group,    -   chlorosulfonyl (—SO₂Cl) group,    -   a —O—C(═O)—C(N₂)—CF₃ group or a —C(═O)—C(N₂)—CF₃ group,    -   activated esters, such as —C(═O)—NHS, wherein NHS stands for        N-hydrosuccinimidyl, perfluorinated esters, and acylureas,    -   a —C≡C—C≡N group,    -   tags, and    -   alkyl groups substituted by at least one of the previously        listed groups,

wherein at least one of R₁ to R₅ comprises, preferably is, a tag moiety.

Two of R₁ to R₅ may alternatively form together and with the carbonatoms of the phenyl ring to which they are linked a mono or polycyclicring, saturated, unsaturated or aromatic, optionally comprising at leastone heteroatom such as P, O or S.

The tag moiety that is comprised in the compound of formula (I) may bedirectly bonded to the phenyl ring. It may also be bonded to the phenylring through a “linker” group, such as a COO, a NH—C(═O)—NH, aNH—C(═O)—O, a triazole, or a CONH group. It may also be present as asubstituent of one of the R₁ to R₅ groups as described above. In apreferred embodiment, R₃ comprises, preferably R₃ is, a tag moiety.

In the present invention, the term “alkyl” relates to a linear, cyclicor branched hydrocarbon group comprising from 1 to 20 carbon atoms,preferably from 1 to 6 carbon atoms, in particular from 1 to 3 carbonatoms. Among alkyl groups can be cited for instance the methyl, ethyl,n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, n-hexyland cyclohexyl groups. An alkyl group according to the invention may beinterrupted by at least one heteroatom selected from Si, N, O and S.Among alkyl groups interrupted by at least one heteroatom may be citedthe polyethyleneglycol groups of formula —(OCH₂—CH₂)_(n)—OH, wherein nis from 1 to 1000, preferably from 1 to 100, in particular from 1 to 8.An alkyl group according to the invention may be substituted by at leastone halogen atom.

The term “alkene” relates to an alkyl group as defined above, furthercomprising at least one C═C double bond.

The term “alkyne” relates to an alkyl group as defined above, furthercomprising at least one C≡C triple bond. Among alkyne groups can becited for instance acetylene and cyclooctyne groups.

The term “alkoxy” relates to an alkyl group as defined above linked tothe rest of the molecule via an oxygen atom.

The term “aryl” relates to a group comprising at least one planar ringcomprising a conjugated π system made of double bonds and/or non-bondingdoublets, wherein each atom of the ring comprises a p orbital, the porbitals overlay each other, and the delocalization of the π electronslowers the molecule energy. Preferably, the aryl group is a hydrocarbonaryl group, optionally comprising at least one heteroatom selected fromN, O and S. Preferably, an aryl group is selected from the groupconsisting of phenyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl,triazinyl, furanyl, thiophenyl, pyrrolyl, imidazolyl, thiazolyl,oxazolyl, isoxazolyl, dihydroisoxasolyl, triazolyl, diazinyl,tetrazinyl, pyrazolyl and naphthyl groups. In particular, an aryl groupis selected from the group consisting of isoxazolyl, dihydroisoxasolyl,triazolyl, diazinyl, tetrazinyl and pyrazolyl groups.

The term “halogen” relates to an atom selected from the group consistingof F, Cl, Br and I atoms. Preferably, a halogen is a Cl or Br atom.

The optical and geometrical isomers, racemates, tautomers, salts,hydrates, solvates and mixtures thereof of the compounds are alsoencompassed by the scope of formulas (I), (II), (III) and (IV) of thepresent invention.

When the compounds according to the invention are in the forms of salts,they are preferably pharmaceutically acceptable salts. Such saltsinclude pharmaceutically acceptable acid addition salts,pharmaceutically acceptable base addition salts, pharmaceuticallyacceptable metal salts, ammonium and alkylated ammonium salts. Acidaddition salts include salts of inorganic acids as well as organicacids. Representative examples of suitable inorganic acids includehydrochloric, hydrobromic, hydroiodic, phosphoric, sulfuric, nitricacids and the like. Representative examples of suitable organic acidsinclude formic, acetic, trichloroacetic, trifluoroacetic, propionic,benzoic, cinnamic, citric, fumaric, glycolic, lactic, maleic, malic,malonic, mandelic, oxalic, picric, pyruvic, salicylic, succinic,methanesulfonic, ethanesulfonic, tartaric, ascorbic, pamoic,bismethylene salicylic, ethanedisulfonic, gluconic, citraconic,aspartic, stearic, palmitic, EDTA, glycolic, p-aminobenzoic, glutamic,benzenesulfonic, p-toluenesulfonic acids, sulphates, nitrates,phosphates, perchlorates, borates, acetates, benzoates,hydroxynaphthoates, glycerophosphates, ketoglutarates and the like.Further examples of pharmaceutically acceptable inorganic or organicacid addition salts include the pharmaceutically acceptable salts listedin J. Pharm. Sci. 1977, 66, 2. Preferably, the salt does not compriseany thiol moiety.

The “counterion” can be any ion appropriate for compensating the chargeof the diazonium group, and may be easily chosen by anyone of ordinaryskill in the art. For instance, the counterion may be selected from thegroup consisting of halogenates, BF₄ ⁻, NO₃ ⁻, HSO₄ ⁻, PF₆ ⁻ CH₃COO⁻,N(SO₂CF₃)₂ ⁻, CF₃SO₃ ⁻, CH₃SO₃ ⁻, CF₃COO⁻, (CH₃O)(H)PO₂ ⁻ and N(CN)₂ ⁻.

In the present invention, the hydroxyl (OH), amino (NH₂ or NHR) andcarboxyl (COOH) groups may be protected with appropriate protectinggroups. One can refer to T. W. Green, P. G. M. Wuts, Protective Groupsin Organic Synthesis, Wiley-Interscience, New York, 1999.

Among protecting groups for hydroxyl groups may be cited acetyl (Ac),benzoyl (Bz), benzyl (Bn), β-Methoxyethoxymethyl ether (MEM),Dimethoxytrityl, [bis-(4-methoxyphenyl)phenylmethyl] (DMT),Methoxymethyl ether (MOM), Methoxytrityl[(4-methoxyphenyl)diphenylmethyl, MMT), p-Methoxybenzyl ether (PMB),Methylthiomethyl ether, Pivaloyl (Piv), Tetrahydropyranyl (THP),Tetrahydrofuran (THF), and Trityl (triphenylmethyl, Tr).

Among protecting groups for amino groups may be cited t-butyl carbamate(Boc), 2-trimethylsilylethyl carbamate (Teoc),1-(1-Adamantyl)-1-methylethyl carbamate (Adpoc),1-Methyl-1-(4-biphenyl)ethyl carbamate (Bpoc),1-(3,5-Di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 1-AdamantylCarbamate (Adoc), p-Methoxybenzyl carbamate (Moz), 9-Anthrylmethylcarbamate, Diphenylmethyl Carbamate, 9-Fluorenylmethyl carbamate (Fmoc),9-(2-Sulfo)Fluoroenylmethyl carbamate, dibromo)Fluorenylmethylcarbamate,2,7-Di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methylcarbamate(DBD-Tmoc), 2-(N,N-dicyclohexylcarboxamido)ethylCarbamate,2-Phosphonioethyl carbamate (Peoc), 2-Phenylethyl carbamate, Benzylcarbamate (Cbz), Allyl carbamate (Alloc), 1-Isopropylallyl carbamate(Ipaoc), 4-Nitrocinnamyl Carbamate (Noc), 8-Quinolyl carbamate andN-Phtalimide.

Among protecting groups for carboxyl groups may be cited methyl esters,benzyl esters, tert-butyl esters, silyl esters, 2,6-dimethylphenol,2,6-diisopropylphenol, and 2,6-di-tert-butylphenol.

The term “tag” or “tag moiety” relates to a chemical group appropriatefor allowing one or several of the following:

-   -   detection of the compound,    -   vectorization of an agent of interest by the compound,    -   solubilization of the compound,    -   stabilization of the compound,    -   improvement of the extraction and/or purification of the        compound,    -   modification of at least one of the ADME (Administration        Distribution Metabolisation Excretion) parameters of the        compound;    -   addition of bioactivity to the compound;    -   addition of an appropriate functionality for click-chemistry.

The compounds comprising such tags and according to the invention cantherefore be used as a tool for detection, vectorization of an agent ofinterest, solubilization, stabilization, improvement of the extractionand/or purification, modification of at least one of the ADME(Administration Distribution Metabolisation Excretion) parameters;addition of bioactivity; and/or addition of an appropriate functionalityfor click-chemistry.

A chemical group appropriate for allowing detection of the compound ofthe invention may be any chemical group that can be identified and/orquantified by any technique of analysis known in the art. Among tags fordetection can be cited fluorescent, such as fluorescent probes, such asfluorescein, quantum dots, cyanine dyes Cy3® and Cy5®, Alexa Fluor®dyes, Dylight Fluor® dyes, IRIS® Dyes, Seta® dyes, SeTau® dyes, SRfluor®dyes, Square® dyes, Nile red,

or carboxytetramethylrhodamine (TAMRA); Nuclear Magnetic Resonance (NMR)tags, such as xenon or lanthanides (in particular terbium Tb or europiumEu); magnetic resonance imaging (MRI) contrast agents such as Gdchelates; mass spectrometry tags such astris(2,4,6-trimethoxyphenyl)phosphonium (TMPP) or isotope-coded tags;infrared (IR) tags; positron emission tomography (PET) tags;single-photon emission computed tomography (SPECT) tags; tritium ordeuterium atoms; microscopy tags such as gold nanoparticles; quencherssuch as dabsyl (dimethylaminoazobenzene sulfonic acid).

A chemical group appropriate for allowing vectorization of an agent ofinterest with the compound of the invention may be any chemical group orbiological moiety appropriate for helping the compound and/or the agentof interest to reach the appropriate tissue or organ, such as liver orbladder. For instance, the vectorization tag may be a chemical groupable to form micelles, reverse-micelles or liposomes, such as anamphiphilic chemical group, a nano or microparticle, a viral vector, apolymer, a folate, an ammonium group, a peptide, an EGFR (EpidermalGrowth Factor Receptor) ligand or an antibody.

A chemical group appropriate for allowing stabilization of the compoundis a chemical group that affords increasing the half-life of thecompound, preferably in vivo. For instance, the stabilization tag may bealbumin, such as Human Serum Albumin HSA or Bovine Serum Albumin BSA.

A chemical group appropriate for allowing modification of the ADMEparameters of the compound can be for instance a therapeutic agent, adrug, a prodrug, a polyethylene glycol of formula —(OCH₂—CH₂)_(n)—OR″,wherein R″ is a hydrogen atom or an alkyl group, wherein n is from 1 to1000, preferably from 1 to 100, in particular from 1 to 8, a peptide,such as Proline-Alanine-Serine (Pro-Ala-Ser) or poly-Glu, a polypeptide,such as XTEN recombinant polypeptide, a lipid, such as palmitic acid, acarbohydrate, hydroxyethyl starch, a nucleic acid, such as DNA or RNA,in particular siRNA. The term “prodrug” relates to a variant of a drugthat can be transformed in vivo into a drug. The term “peptide” relatesto a peptide comprising from 1 to 20, preferably from 1 to 10,aminoacids.

The selection of the appropriate tag, such as the determination of thenumber of ethylene glycol moieties, can be easily adjusted by one ofordinary skill in the art depending of the desired ADME modification.

A chemical group appropriate for allowing extraction and/or purificationof the compound may be any chemical group that favors and/or facilitatesthe extraction and/or purification of the compound of the invention.Among extraction and/or purification-tags can be cited biotin, chelatingtags such as DTPA (diethylenetriaminepentaacetic acid), EDTA(ethylenediamine-N,N,N′,N′-tetraacetic acid), NTA (nitrilotriaceticacid) and D4 (octamethylcyclotetrasiloxane), protein tags such aspolyarginine or polyhistidine tags, preferably His6 or His10 tags,FLAG-tag, Strep-tag, c-myc-tag, S-tag, calmodulin-binding peptide,cellulose-binding domain, SBP-tag, chitin-binding domain, glutathioneS-transferase tag, maltose-binding protein, NusA, TrxA and DsbA tags,boronic tags such as[(3-oxospiro[isobenzofuran-1(3H),9′-[9H]-flxanthene]-3′,6′-diyl)bis(iminomethylene-2,1-phenylene)]bis-(9Cl),perfluoroalkyl groups, ionic (cationic or anionic) groups, such asammonium groups, and solid surfaces such as polymeric materials, inparticular polyethylene beads, nanoparticles, in particular magneticnanoparticles, chips, silica beads or silica wafers.

A chemical group appropriate for addition of bioactivity may be forinstance a chemical group comprising at least one radioisotope, such as¹³¹I, ⁹⁰Y, ⁸⁹Sr, or ¹⁵³Sm, or a derivative thereof.

A chemical group appropriate for reacting in click-chemistry may be forinstance a chemical group selected from the group consisting of azides(such as N₃) and strained alkynes, in particular cyclic alkynes. Amongcyclic alkynes may be cited for instance the bicyclononyne (BCN) andtetramethylthiepinium (TMTI) moieties.

In an embodiment, each of R₁ to R₅ is independently selected in thegroup consisting of:

-   -   alkyne groups,    -   amino groups,    -   hydroxylamine (—ONH₂) groups,    -   hydrazine (—NH—NH₂) groups,    -   azido (N₃) groups,    -   diazonium (N₂ ⁺) groups, preferably in presence of a counterion,    -   maleimide groups,    -   carboxylic acid groups,    -   aldehyde (—CHO) groups,    -   boronic —B(OR″)₂ groups, wherein R″ is as described above, and    -   activated esters.

In a preferred embodiment, none of R₁ to R₅ comprises a free SH group.In a preferred embodiment, R₁ and/or R₅ do not comprise any nucleophilicgroup, such as amino groups, hydroxylamine groups, hydrazine groups orhydroxyl groups.

In specific embodiments, 1, 2, 3, 4 or 5 of R₁ to R₅ are different fromhydrogen atoms.

In specific embodiments, at least one of R₁ to R₅ comprises at least onemoiety that is appropriate for further forming a covalent bond with achemical group selected in the group consisting of thiol (SH) moieties,amine (NH₂) moieties and carboxylic acid (COOH) moieties. Among moietiesappropriate for forming a covalent bond with thiol moieties, one cancite maleimide moieties. Among moieties appropriate for forming acovalent bond with amine moieties, one can cite NHS-ester moieties.

Among compounds of formula (I) according to the present invention may becited the following compounds:

Among compounds of formula (I) may also be cited the followingcompounds:

Synthesis of Compounds of the Invention

Compounds of formula (I) or (II) according to the invention can besynthesized for instance in two steps from the corresponding iodoarene,by coupling with propargyl alcohol, for instance via a Sonogashiracoupling. The coupling is preferably followed by oxidation; forinstance, the oxidation may be a tandem oxidation performed with MnO₂ inthe presence of an ammonia solution. Alternatively, the compounds of theinvention can be synthesized by cyanation of arylalkynes. Cyanation maybe performed for instance with CuCN, arylisocyanates,cyanobenzotriazoles or cyanoimidazoles.

Labeling of Compounds Comprising at Least One Thiol Moiety

The compounds of formula (I) may be used in a process for labelingcompounds comprising at least one thiol SH moiety. Preferably, theprocess of the invention comprises contacting at least one compound offormula (I) with a compound comprising at least one thiol SH moiety, ora sample susceptible to comprise such a compound. Preferably, the sampleis a biological sample, in particular an aqueous sample.

The term “labeling” in the present invention refers to the formation ofa covalent bond between the sulfur atom of the thiol moiety and thepropiolonitrile moiety of the compounds of formula (I).

The compound comprising a thiol moiety (R₆—SH compound) can be forinstance a fluorophore, a quencher, an amino acid, a peptide, a protein,an enzyme, a drug, a prodrug and/or a drug metabolite.

R₆ may be any chemical group that is bonded to a thiol (SH) group toform a “compound comprising a thiol moiety”. R₆ preferably comprisescarbon, hydrogen, oxygen, nitrogen, phosphorus, and/or sulphur atoms.

In particular, the compound comprising a thiol moiety can be cystein, ora derivative, such as an ester, thereof, or a peptide or a proteincomprising at least one cystein residue. Alternatively, the compoundcomprising a thiol moiety may be a surface presenting at least one freeSH group.

Labeling of the compounds comprising a thiol moiety with compounds offormula (I) may be used for a great number of applications.

In a first embodiment, labeling of the compounds comprising a thiolmoiety with compounds of formula (I) may be used in the detection and/orquantification of the compound comprising the thiol moiety in a sample.The detection means in the present invention identifying the presence orabsence of the desired compound(s) in the sample. The sample can be anysample susceptible to comprise the compound comprising at least onethiol moiety. For instance, the sample may be a biological sample, forinstance a biological fluid, such as blood, plasma, serum, saliva,urine, etc., an extract of natural products, a biological tissue, or apart thereof, or a medium comprising cells.

In a second embodiment, labeling of the compounds comprising a thiolmoiety with compounds of formula (I) may be used for conjugation of thecompound comprising a thiol moiety with a moiety that improves itsphysico-chemical properties. For instance, the conjugated moiety mayimprove the solubility of the compound comprising a thiol moiety, orimprove its synthesis and/or purification.

In a third embodiment, labeling of the compounds comprising a thiolmoiety with compounds of formula (I) may be used for bio-conjugation ofthe compound comprising a thiol moiety with a compound of interest, suchas a drug, a prodrug, a carbohydrate or a protein.

For instance, a compound of formula (I) comprising a compound ofinterest as tag may allow selective vectorization and/or binding of thecompound of interest to the compound comprising at least one thiolmoiety.

Another object of the invention is a compound of formula (I)

as defined above, including the described specific embodiments.

The invention also discloses a compound of formula (II)

wherein each of R₁ to R₅ is selected independently in the groupconsisting of:

-   -   hydrogen atoms,    -   alkyl, alkene or alkyne groups, optionally interrupted by at        least one heteroatom selected among O, N and S,    -   aryl groups,    -   alkoxy groups,    -   halogen atoms,    -   amino (—NRR′) groups, wherein R and R′ are independently        hydrogen atoms or alkyl, alkene, alkyne or aryl groups as        defined above,    -   hydroxylamine (—ONH₂) group,    -   hydrazine (—NH—NH₂) group,    -   nitro (—NO₂) group,    -   azido (—N₃) group,    -   diazonium (—N₂ ⁺) group, preferably in presence of a counterion,    -   maleimide group,    -   alkyl- or aryl-carboxyl (—C(═O)OR) groups,    -   alkyl- or aryl-carbonyl (—C(═O)R) groups,    -   hydroxyl (—OH) group,    -   boronic —B(OR″)₂ group, wherein R″ is a hydrogen atom or an        alkyl group,    -   phosphine or phosphonium groups,    -   isocyanate (—N═C═O) or isothiocyanate (—N═C═S) group,    -   chlorosulfonyl (—SO₂Cl) group,    -   a O—C(═O)—C(N₂)—CF₃ group or a —C(═O)—C(N₂)_CF₃ group,    -   activated esters, such as —C(═O)—NHS, perfluorinated esters and        acylureas,    -   tags, and    -   alkyl groups substituted by at least one of the previously        listed groups.

The compounds of formula (II) are linkers to which at least one tag maybe added to form the compounds of formula (I) as described above.

Among the compounds of formula (II) according to the present inventionmay be cited the following compounds:

Among the compounds of formula (II) according to the present inventionmay also be cited the following compounds:

The process for labeling a compound comprising a thiol moiety accordingto the invention may further comprise, before the step of contacting thecompound comprising a thiol moiety with a compound of formula (I), apreliminary step of preparation of the compound of formula (I),comprising contacting a compound of formula (II) with a compoundcomprising a tag moiety, or a precursor thereof.

The term “precursor” relates in the present case to a chemical groupthat is able to form, after contacting with the compound of formula(II), the tag moiety.

Preferably, at least one of R₁ to R₅ is different from a hydrogen atom.

An object of the invention is a compound of formula (II), wherein atleast one of R₁ to R₅ comprises, preferably is, a maleimide, an azide(N₃) group, an alkyne or a NHS-ester moiety.

The maleimide and NHS-ester moieties respectively allow further linkingof the compound to another thiol or an amine group; the N₃ group allowsfurther linking of the compound to another alkyne-group and the alkynegroup allows further linking of the compound to another N₃ group.

In an embodiment, the compound of formula (II) according to theinvention is selected from the following compounds:

The invention also relates to compounds of formula (I) or (II), whereinat least one of R₁ to R₅ is further bonded to a compound of interest.

A compound of interest may be for instance a molecule, such as afluorophore, for instance rhodamine, a group of atoms comprising atleast one radioactive atom (¹⁴C, ³H, or ¹³¹I for instance), a group ofatoms of known mass (a mass tag), a ligand, a drug, a therapeutic agent,a biomolecule, such as an antibody, a protein, such as BSA (bovine serumalbumin), a DNA fragment, a nanoobject, such as a nanoparticle (ie anobject or a particle of 0.1 to 1000 nm), or a support, such as apolymer.

When the process of labeling according to the invention is performedwith such a compound of formula (I) or (II) wherein at least one of R₁to R₅ is further bonded to a compound of interest, the process affordsthe formation of a conjugate between the compound of interest and thecompound comprising a thiol moiety.

In an embodiment, the compound of interest is a biomolecule such as aprotein or an antibody, and the compound comprising a thiol moiety is afluorophore such as a compound comprising a TAMRA moiety and a thiolmoiety (TAMRA-SH).

In an embodiment, the compound of interest is a biomolecule such as aprotein or an antibody, and the compound comprising a thiol moiety is adrug or a therapeutic agent. The conjugate obtained by the process oflabeling according to the invention is a therapeutic antibody.

Another object of the invention is a compound of formula (III):

wherein R₆—S corresponds to the moiety of the compound comprising atleast one thiol moiety as defined above. In particular, the compound offormula (III) is of formula (IV):

wherein R₇ and R₅ are selected in the group consisting of OH, tag,alkyl, O-alkyl, and peptidic moieties, wherein the alkyl groups may besubstituted by at least one tag moiety. A peptidic moiety is a moietycomprising at least one aminoacid, when the moiety presents more thanone aminoacid (2, 3, 4, 5, . . . ), the aminoacids are linked betweeneach other by peptidic bonds. Preferably, the double bond of thecompound of formula (III) or (IV) is of (Z) configuration. The tag andalkyl groups are as defined above.

Among compounds of formula (III) according to the present invention maybe cited the following compounds:

The compounds of formula (I) have surprisingly been found to be morestable in aqueous medium than the corresponding compounds wherein the3-arylpropiolonitrile moiety is replaced with a maleimide moiety, whichis classically used for labeling thiol moieties. For instance, compound1

was approximately 25% hydrolyzed after 1 h in buffer solution(k_(obs)=7×10⁻⁵ s⁻¹), while no hydrolysis could be detected for compound11

Interestingly, even after a week at room temperature, no trace ofhydrolyzed product could be detected for compound 11.

In addition, the compounds of formula (II) according to the inventionshowed a marked selectivity towards cysteine compared to otheramino-acids which do not comprise free thiol moieties. Comparatively,the chemoselectivity obtained for the corresponding compounds whereinthe 3-arylpropiolonitrile moiety is replaced with a maleimide moiety islower.

Finally, the compounds of formula (III) as described above have shown tobe highly more stable in biological conditions than the correspondingcompounds wherein the 3-arylpropiolonitrile moiety is replaced with amaleimide moiety. For instance, the addition product

between compound

and cysteine derivative 3

was particularly stable in a wide range of conditions, such asphysiological conditions. In particular, said addition product wasstable in a wide range of pH (from 0 to 14), and no exchange productcould be observed after one hour of reaction with an excess ofphenylthiol and glutathione.

The invention will be illustrated in more detail with reference to thefollowing examples, but it should be understood that the presentinvention is not deemed to be limited thereto.

EXAMPLES Example 1 Synthesis of Compounds of the Invention

Synthesis of Compounds of Formula (II)

A series of compounds of formula (I) or (II) were synthesized andcharacterized according to the following procedures.

General Protocols:

Sonogashira Coupling

Standard Reaction Protocols:

A. To a degassed solution of the proper aryl halide (1 eq., 1 mmol) inDMF (5 mL) and DIPEA (10 eq., 10 mmol), premixed PdCl₂(PPh₃)₂ (0.03 eq.,30 μmol) and CuI (0.06 eq., 60 μmol) were added. Obtained reaction masswas degassed, stirred for another 5 minutes, followed by the addition ofpropargyl alcohol (1.2 eq., 1.2 mmol). The reaction mass was stirred for1-24 hours (monitored by TLC). 1M HCl (50 mL) was added (if containsfree amino groups, 50 mL of water were added instead) and the reactionmixture was extracted with ethyl acetate (3×20 mL). United ethyl acetatefractions were washed with water (1×10 mL) dried over MgSO₄ andevaporated to give crude product. Products were purified by flashchromatography (gradient of 20 minutes from 100% of cyclohexane to 100%of ethyl acetate).

B. To a degassed solution of the proper aryl halide (1 eq., 1 mmol) inTHF (5 mL) and TEA (5 mL), premixed PdCl₂(PPh₃)₂ (0.03 eq., 30 μmol) andCuI (0.06 eq., 60 μmol) were added, followed by the addition ofpropargyl alcohol (2 eq., 1.2 mmol). The reaction mass was stirred for1-24 hours (monitored by TLC). THF and TEA were evaporated and the crudeproduct was purified by flash chromatography (gradient of 20 minutesfrom 100% of cyclohexane to 100% of ethyl acetate).

C. To a degassed solution of the proper aryl halide (1 eq., 1 mmol) inpropylamine or pyrrolidine (3 mL), Pd(PPh₃)₄ (0.05 eq., 50 μmol) wasadded. The reaction mass was heated overnight (30-50° C.), evaporatedand the crude product was purified by flash chromatography (gradient of20 minutes from 100% of cyclohexane to 100% of ethyl acetate).

Preparation of Highly Active MnO₂

A solution of MnCl₂.4H₂O (1 eq., 1 mole, 200 g) in water (2 L) at 70° C.was gradually added during 10 minutes, with stirring, to a solution ofKMnO₄ (1 eq., 1 mole, 160 g) in water (2 L) at 60° C. in a hood. Avigorous reaction ensued with evolution of chlorine; the suspension wasstirred for 2 hours and kept overnight at room temperature. Theprecipitate was filtered off, washed thoroughly with water (4 L) untilpH 6.5-7 and the washing gave a negligible chloride test. The filtercake was then dried at 120-130° C. for 18 h; this gave achocolate-brown, highly disperse amorphous powder.

MnO₂ Oxidation

Slightly modified procedure described by McAllister et al.⁸²⁴ To asolution of the proper propargylic alcohol (1 eq., 1 mmol) in THF (4.5mL), MgSO₄ (15 eq., 15 mmol), highly active MnO₂ (25 eq., 25 mmol) and2M NH₃ solution in IPA (4 eq., 4 mmol, 2 mL) were added. Obtainedreaction mass was vigorously stirred at room temperature for 0.5-12hours (monitored by TLC). DCM (20 mL) were added and the obtainedreaction mass was filtered through Celite, evaporated to give crudeproduct and purified by flash chromatography if required.

Substituted 3-(aryl)prop-2-yn-1-ols (1a-12a)

R₁ R₂ R₃ R₄ X Protocol  1a OMe H H H I B  2a H OMe H H Br C  3a H H OMeH I B  4a OMe H H OMe I C  5a NH₂ H H H I B  6a H NH₂ H H I A  7a H HNH₂ H I A  8a Me H H H I B  9a Me H H Me I A 10a NO₂ H H H Br B 11a H HNHAc H I A 12a H H CONHMe H I A

3-(2-Methoxyphenyl)prop-2-yn-1-ol (1a)

Reaction time: 18 hours; yield: 72%.

¹H NMR (400 MHz, METHANOL-d₄) δ 7.36 (dd, J=1.5, 7.5 Hz, 1H), 7.26-7.33(m, 1H), 6.97 (d, J=8.3 Hz, 1H), 6.86-6.93 (m, 1H), 4.43 (s, 2H), 3.84(s, 3H); ¹³C NMR (101 MHz, METHANOL-d₄) δ 161.6, 134.5, 131.0, 121.5,113.4, 112.0, 92.7, 82.0, 56.2, 51.5.

3-(3-Methoxyphenyl)prop-2-yn-1-ol (2a)

Reaction time: 16 hours; yield: 87%.

¹H NMR (400 MHz, METHANOL-d₄) δ 7.15-7.24 (pseudo-t, J=7.5 Hz, 1H), 7.00(d, J=7.5 Hz, 1H), 6.93-6.98 (m, 1H), 6.87 (dd, J=2.13, 7.5 Hz, 1H),4.41 (s, 2H), 3.73 (s, 3H); ¹³C NMR (101 MHz, METHANOL-d₄) δ 160.9,130.6, 125.4, 125.1, 117.8, 115.7, 88.8, 85.6, 55.9, 51.4.

3-(4-Methoxyphenyl)prop-2-yn-1-ol (3a)

Reaction time: 16 hours; yield: 92%.

¹H NMR (400 MHz, CHLOROFORM-d) δ 7.40 (d, J=8.78 Hz, 2H), 6.86 (d,J=8.78 Hz, 2H), 4.51 (d, J=4.9 Hz, 2H), 3.83 (s, 3H), 1.78 (t, J=4.9 Hz,1H); ¹³C NMR (101 MHz, CHLOROFORM-d) δ 159.8, 133.2, 114.6, 114.0, 85.9,85.7, 55.3, 51.7.

3-(2-Aminophenyl)prop-2-yn-1-ol (4a)

Reaction time: 24 hours; yield: 62%.

¹H NMR (400 MHz, METHANOL-d₄) δ 7.19 (dd, J=1.25, 7.9 Hz, 1H), 7.03-7.12(m, 1H), 6.75 (d, J=7.9 Hz, 1H), 6.56-6.65 (m, 1H), 4.47 (s, 2H), 4.26(s, 2H); ¹³C NMR (101 MHz, METHANOL-d₄) δ 150.3, 133.0, 130.6, 118.2,115.6, 93.8, 78.9, 69.5, 51.0.

3-(3-Aminophenyl)prop-2-yn-1-ol (5a)

Reaction time: 18 hours; yield: 77%.

¹H NMR (400 MHz, CHLOROFORM-d) δ 7.08 (t, J=7.8 Hz, 1H), 6.83 (d, J=7.8Hz, 1H), 6.76 (s, 1H), 6.64 (dd, J=1.5, 7.8 Hz, 1H), 4.46 (s, 2H), 2.17(s, 1H); ¹³C NMR (101 MHz, CHLOROFORM-d) δ 146.3, 129.2, 123.4, 122.0,118.0, 115.5, 87.0, 85.6, 51.4.

3-(4-Aminophenyl)prop-2-yn-1-ol (6a)

Reaction time: 18 hours; yield: 42%.

¹H NMR (400 MHz, METHANOL-d₄) δ 7.11-7.21 (d, J=8.5 Hz, 2H), 6.53-6.68(d, J=8.5 Hz, 2H), 4.37 (s, 2H); ¹³C NMR (101 MHz, METHANOL-d₄) δ 149.7,133.8, 115.7, 112.5, 86.7, 85.9, 51.4;

3-(2-Nitrophenyl)prop-2-yn-1-ol (7a)

Reaction time: 15 hours; yield: 35%.

¹H NMR (400 MHz, CHLOROFORM-d) δ 7.97 (d, J=8.0 Hz, 1H), 7.54-7.60 (d,J=8.0 Hz, 1H), 7.46-7.54 (t, J=8.0 Hz, 1H), 7.36-7.44 (t, J=8.0 Hz, 1H),4.49 (s, 2H), 1.68 (br. s., 1H); ¹³C NMR (101 MHz, CHLOROFORM-d) δ149.9, 134.8, 132.8, 128.9, 124.6, 118.0, 95.2, 80.9, 51.7.

4-(3-Hydroxyprop-1-yn-1-yl)-N-methylbenzamide (8a)

Reaction time: 12 hours; yield: 91%.

¹H NMR (400 MHz, METHANOL-d₄) δ 7.72-7.82 (m, J=8.28 Hz, 2H), 7.41-7.53(m, J=8.28 Hz, 2H), 4.43 (s, 2H), 2.92 (s, 3H); ¹³C NMR (101 MHz,METHANOL-d₄) δ 169.9, 135.2, 132.7, 128.3, 127.6, 91.5, 84.7, 51.3,27.1.

N-(4-(3-Hydroxyprop-1-yn-1-yl)phenyl)acetamide (9a)

Reaction time: 18 hours; yield: 85%.

¹H NMR (400 MHz, METHANOL-d₄) δ 7.56-7.64 (d, J=8.8 Hz, 2H), 7.47-7.56(d, J=8.8 Hz, 2H), 4.74 (s, 2H), 2.04 (s, 3H); ¹³C NMR (101 MHz,METHANOL-d₄) δ 171.9, 143.8, 135.7, 120.7, 112.8, 106.2, 84.5, 62.7,24.1.

3-(o-Tolyl)prop-2-yn-1-ol (10a)

Reaction time: 5 hours; yield: 70%.

¹H NMR (400 MHz, CHLOROFORM-d) δ 7.40 (d, T=7.5 Hz, 1H), 7.11-7.24 (m,3H), 4.54 (s, 2H), 2.43 (s., 3H); ¹³C NMR (101 MHz, CHLOROFORM-d) δ138.2, 131.2, 128.4, 128.0, 119.3, 115.3, 86.5, 85.2, 51.2, 21.2.

3-(2,6-Dimethylphenyl)prop-2-yn-1-ol (11a)

Reaction time: 24 hours; yield: 25%.

¹H NMR (400 MHz, CHLOROFORM-d) δ 7.03 (t, J=7.5 Hz, 1H), 6.95 (d, J=7.5Hz, 2H), 4.50 (s, 2H), 2.34 (s, 7H); ¹³C NMR (101 MHz, CHLOROFORM-d) δ140.5, 127.9, 126.7, 122.3, 95.6, 83.3, 51.9, 21.1.

3-(2,6-Dimethoxyphenyl)prop-2-yn-1-ol (12a)

Reaction conditions: 30° C., propylamine, 16 hours; yield: 38%.

¹H NMR (400 MHz, METHANOL-d₄) δ 7.25 (t, J=8.4 Hz, 1H), 6.62 (d, J=8.4Hz, 2H), 4.46 (s, 2H), 3.84 (s, 6H); ¹³C NMR (101 MHz, METHANOL-d₄) δ163.0, 131.0, 104.7, 102.5, 97.0, 78.1, 56.4, 51.7.

Substituted 3-aryl-propiolonitriles (1-12)

R₁ R₂ R₃ R₄ Time, h Yield, %  1 OMe H H H 3 45  2 H OMe H H 2 85  3 H HOMe H 3 95  4 OMe H H OMe 4 60  5 NH₂ H H H 1 47  6 H NH₂ H H 2 71  7 HH NH₂ H 9 94  8 Me H H H 1.5 70  9 Me H H Me 2 55 10 NO₂ H H H 4 21 11 HH NHAc H 2 92 12 H H CONHMe H 2 61

3-(2-Methoxyphenyl)propiolonitrile (1, APN-o-OMe)

¹H NMR (400 MHz, METHANOL-d₄) δ 7.51-7.65 (m, 2H), 7.12 (d, J=8.3 Hz,1H), 7.01 (t, J=7.7 Hz, 1H), 3.94 (s, 4H); ¹³C NMR (101 MHz,METHANOL-d₄) δ 164.7, 136.4, 135.3, 122.0, 112.6, 107.7, 106.4, 81.8,66.7, 56.7; IR (neat film, cm⁻¹): 2946, 2264, 2142 1596, 1490, 1245,1164, 1122, 1047, 1021, 752, 498; GC-ESI-HRMS: 157.05276; found157.05044.

3-(3-Methoxyphenyl)propiolonitrile (2, APN-m-OMe)

¹H NMR (400 MHz, METHANOL-d₄) δ 7.38 (t, J=7.8 Hz, 1H), 7.27 (d, J=7.8Hz, 1H), 7.20-7.24 (m, 1H), 7.12-7.20 (m, 1H), 3.83 (s, 3H); ¹³C NMR(101 MHz, METHANOL-d₄) δ 161.2, 131.4, 127.1, 120.0, 119.4, 119.2,106.0, 84.1, 62.7, 56.1; IR (neat film, cm⁻¹): 2491, 2264, 2144, 1595,1573, 1488, 1464, 1420, 1324, 1294, 1207, 1178, 1045, 783, 681, 494;GC-ESI-HRMS: 157.05276; found 157.05298.

3-(4-Methoxyphenyl)propiolonitrile (3, APN-p-OMe)

¹H NMR (400 MHz, CHLOROFORM-d) δ 7.46-7.70 (m, J=8.8 Hz, 2H), 6.86-6.96(m, J=8.8 Hz, 2H), 3.86 (s, 3H); ¹³C NMR (101 MHz, CHLOROFORM-d) δ161.4, 134.4, 113.7, 108.2, 104.8, 82.7, 61.5, 54.5; IR (neat film,cm⁻¹): 2985, 2358, 2342, 2263, 2178, 2149, 1603, 1514, 1307, 1270, 1180,1028, 835, 808, 669, 424; GC-ESI-HRMS: 157.05276; found 157.05337.

3-(2,6-Dimethoxyphenyl)propiolonitrile (4, APN-o,o′-diOMe)

¹H NMR (400 MHz, CHLOROFORM-d) δ 7.38 (t, J=8.5 Hz, 1H), 6.53 (d, J=8.5Hz, 2H), 3.88 (s, 6H); ¹³C NMR (101 MHz, CHLOROFORM-d) δ 164.4, 133.8,106.2, 103.4, 96.5, 77.7, 70.5, 56.2; IR (neat film, cm⁻¹): 2847, 2359,2259, 2201, 2139, 1926, 1586, 1574, 1478, 1432, 1302, 1255, 1188, 1109,1025, 778, 727, 648, 632, 545, 506, 488, 420; GC-ESI-HRMS: 187.06333;found 184.06465.

3-(2-Aminophenyl)propiolonitrile (5, APN-o-NH₂)

¹H NMR (500 MHz, METHANOL-d₄) δ 6.81 (d, J=7.88 Hz, 1H), 6.65-6.76 (m,1H), 6.08-6.19 (m, 2H), 3.85 (br. s., 1H); ¹³C NMR (126 MHz,CHLOROFORM-d) δ 151.4, 134.0, 133.4, 118.2, 115.0, 105.8, 101.0, 81.6,68.5; IR (neat film, cm⁻¹): 3413, 3332, 3211, 2925, 2853, 2250, 2136,1632, 1600, 1563, 1486, 1452, 1312, 1273, 1252, 1161, 740, 673, 493;GC-ESI-HRMS: 142.05310; found 142.05458.

3-(3-Aminophenyl)propiolonitrile (6, APN-m-NH₂)

¹H NMR (400 MHz, CHLOROFORM-d) δ 7.17 (t, J=7.6 Hz, 1H), 6.99 (d, J=7.6Hz, 1H), 6.74-6.89 (m, 2H), 3.85 (br. s., 2H); ¹³C NMR (101 MHz,CHLOROFORM-d) δ 146.8, 129.8, 123.6, 118.7, 118.7, 118.0, 105.7, 83.7,62.3; IR (neat film, cm⁻¹): 3426, 3340, 2923, 2852, 2265, 2142, 1630,1594, 1579, 1513, 1448, 1326, 1313, 1300, 1220, 1164, 993, 882, 862,784, 680, 534, 456; GC-ESI-HRMS: 142.05310; found 142.05197.

3-(4-Aminophenyl)propiolonitrile (7, APN-p-NH₂)

¹H NMR (400 MHz, METHANOL-d₄) δ 7.26 (d, J=8.6 Hz, 2H), 6.51 (d, J=8.6Hz, 2H); ¹³C NMR (101 MHz, METHANOL-d₄) δ 152.5, 135.1, 113.6, 105.6,102.3, 86.3, 60.2; IR (neat film, cm⁻¹): 3431, 3333, 3211, 2250, 2132,1632, 1599, 1513, 1438, 1303, 1178, 1043, 949, 826, 814, 526, 495, 452;GC-ESI-HRMS: 142.05310; found 142.05464.

3-(o-Tolyl)propiolonitrile (8, APN-o-Me)

¹H NMR (400 MHz, CHLOROFORM-d) δ 7.47 (d, J=7.78 Hz, 1H), 7.28-7.36 (m,1H), 7.18 (d, J=8.03 Hz, 1H), 7.08-7.16 (m, 1H), 2.39 (s, 3H); ¹³C NMR(101 MHz, CHLOROFORM-d) δ 143.4, 134.1, 131.8, 130.1, 126.1, 117.4,105.6, 82.4, 66.4, 20.5; IR (neat film, cm⁻¹): 2295, 2257, 2141, 1599,1484, 1456, 1383, 1291, 1199, 1162, 1116, 1039, 757, 711, 672, 548, 490,452; GC-ESI-HRMS: 141.05785; found 141.05926.

3-(2,6-Dimethylphenyl)propiolonitrile (9, APN-o,o′-diMe)

¹H NMR (400 MHz, CHLOROFORM-d) δ 7.12-7.27 (t, J=7.5 Hz, 1H), 7.01 (d,J=7.5 Hz, 2H), 2.38 (s, 6H); ¹³C NMR (101 MHz, CHLOROFORM-d) δ 143.8,131.2, 127.3, 117.6, 105.6, 81.5, 70.2, 20.8; IR (neat film, cm⁻¹):2923, 2856, 2261, 2138, 1732, 1595, 1468, 1381, 1265, 1168, 1033, 774,728, 490; GC-ESI-HRMS: 155.07350; found 155.07507.

3-(2-Nitrophenyl)propiolonitrile (10, APN-o-NO₂)

¹H NMR (400 MHz, METHANOL-d₄) δ 8.28-8.35 (m, 1H), 7.96-8.06 (m, 1H),7.81-7.90 (m, 2H); ¹³C NMR (101 MHz, METHANOL-d₄) δ 151.9, 138.3, 135.2,134.1, 126.6, 114.2, 105.7, 79.0, 68.6; IR (neat film, cm⁻¹): 2268,1604, 1567, 1528, 1502, 1480, 1345, 851, 787, 744, 709, 687, 537, 491;GC-ESI-HRMS: 172.02728; found 172.02869.

N-(4-(Cyanoethynyl)phenyl)acetamide (11, APN-p-NHAc)

¹H NMR (400 MHz, METHANOL-d₄) δ 7.56-7.63 (m, J=8.8 Hz, 2H), 7.49-7.56(m, J=8.8 Hz, 2H), 2.04 (s, 3H); ¹³C NMR (101 MHz, METHANOL-d₄) δ 171.9,143.8, 135.7, 120.7, 112.8, 106.2, 84.5, 62.7, 24.1; IR (neat film,cm⁻¹): 3303, 3174, 3098, 2278, 2262, 2139, 1670, 1594, 1535, 1407, 1364,1321, 1263, 1177, 834, 534; GC-ESI-HRMS: 184.06366; found 184.06212.

4-(Cyanoethynyl)-N-methylbenzamide (12, APN-p-CONHMe)

¹H NMR (400 MHz, METHANOL-d₄) δ 7.96-8.05 (m, J=7.78 Hz, 2H), 7.85-7.93(m, J=7.78 Hz, 2H), 3.03 (s, 3H); ¹³C NMR (101 MHz, METHANOL-d₄) δ169.1, 138.3, 134.9, 129.0, 121.6, 105.9, 83.0, 64.6, 28.8; IR (neatfilm, cm⁻¹): 3348, 2270, 1641, 1549, 1502, 1408, 1392, 1327, 1303, 1283,1162, 854, 760, 617, 488; GC-ESI-HRMS: 184.06366; found 184.06465.

3-(4-Iodophenyl)propiolonitrile (13)

Product was synthesized according to general procedure ofMnO₂-oxidation. Reaction time: 30 minutes; yield: 61%.

¹H NMR (400 MHz, CHLOROFORM-d) δ=7.78 (d, J=8.5 Hz, 2H), 7.32 (d, J=8.5Hz, 3H); ¹³C NMR (101 MHz, chloroform-d) δ 138.1, 134.4, 116.8, 105.2,99.2, 81.9, 64.2.

Compound 13 can be used for the labeling method according to theinvention (with radioisotope 125I).

3-(4-(Trifluoromethyl)phenyl)propiolonitrile (14)

Product was synthesized according to general procedure ofMnO₂-oxidation. Reaction time: 1 hour; yield: 45%.

¹H NMR (400 MHz, CHLOROFORM-d) δ=7.76 (d, J=8.3 Hz, 2H), 7.70 (d, J=8.3Hz, 2H).

Compound 14 can be used for the labeling method according to theinvention (with radioisotope 18F).

tert-Butyl 4-(3-hydroxyprop-1-yn-1-yl)benzoate (15a)

Product was synthesized according to general procedure B for coupling.Yield: 98%.

¹H NMR (400 MHz, CHLOROFORM-d) δ=7.93 (d, J=8.1 Hz, 2H), 7.47 (d, J=8.1Hz, 2H), 4.53 (s, 2H), 1.60 (s, 9H); ¹³C NMR (101 MHz, CHLOROFORM-d)δ=165.1, 131.7, 131.4, 129.3, 126.6, 89.8, 85.1, 81.4, 51.6, 28.1.

tert-Butyl 4-(cyanoethynyl)benzoate (15)

Product was synthesized according to general procedure ofMnO₂-oxidation. Reaction time: 15 minutes; yield: 48%.

¹H NMR (400 MHz, DMSO-d₆) δ=8.00 (d, J=8.3 Hz, 2H), 7.94 (d, J=8.3 Hz,2H), 1.56 (s, 9H); ¹³C NMR (101 MHz, CHLOROFORM-d) δ=164.3, 134.8,133.3, 129.7, 121.3, 105.2, 82.2, 81.9, 64.8, 28.1.

4-(Cyanoethynyl)benzoic acid (16)

To the solution of tert-butyl 4-(2-cyanoeth-1-yn-1-yl)benzoate (1 eq.,350 mg, 1.54 mmol) in MeCN (14 mL) was added TFA (30.6 eq., 5.372 g, 3.5mL, 47.1 mmol). The mixture was stirred for 36 h at r.t. and thenfiltered and washed with 3×2 mL of Et₂O. The precipitate consisted ofpure 4-(2-cyanoeth-1-yn-1-yl)benzoic acid (140 mg, 0.823 mmol, 53%yield).

¹H NMR (400 MHz, METHANOL-d₄) δ=8.12 (d, J=8.3 Hz, 3H), 7.83 (d, J=8.3Hz, 2H).

Perfluorophenyl 4-(cyanoethynyl)benzoate (17)

The solution of pentafluorophenol (1 eq., 89.2 mg, 0.484 mmol) and4-(2-cyanoeth-1-yn-1-yl)benzoic acid (1 eq., 82.9 mg, 0.484 mmol) in THF(4.84 mL) was cooled to 0° C. and DCC (1 eq., 99.9 mg, 0.484 mmol) wasadded to the mixture. The resulting solution was stirred at r.t. for 14h, then filtered and washed with Et₂O. The filtrate was evaporated togive pentafluorophenyl 4-(2-cyanoeth-1-yn-1-yl)benzoate (120 mg, 0.358mmol, 74% yield) as a white solid.

¹H NMR (400 MHz, DMSO-d₆) δ=8.29 (d, J=8.3 Hz, 2H), 8.08 (d, J=8.3 Hz,2H).

Compound 17 can be used for a bio-conjugation method according to theinvention.

Sodium4-((4-(cyanoethynyl)benzoyl)oxy)-2,3,5,6-tetrafluorobenzenesulfonate(18)

To the solution of 4-(2-cyanoeth-1-yn-1-yl)benzoic acid (1 eq., 54.2 mg,0.317 mmol) and sodium 2,3,5,6-tetrafluoro-4-hydroxybenzene-1-sulfonate(1 eq., 84.9 mg, 0.317 mmol) in dry DMF (0.792 mL) was added DCC (1 eq.,65.3 mg, 0.317 mmol). The resulting mixture was stirred at r.t. for 36h, then cooled to 0° C., stirred for 1 h, filtered and washed with 0.8mL of dry DMF. The filtrate was diluted with 16 mL of Et₂O, stirred for15 min for complete crystallization and the precipitate was filtered togive sodium4-((4-(cyanoethynyl)benzoyl)oxy)-2,3,5,6-tetrafluorobenzenesulfonate(72.5 mg, 0.172 mmol, 54% yield) as a white solid.

¹H NMR (400 MHz, DMSO-d₆) δ=8.31 (d, J=6.3 Hz, 2H), 8.09 (d, J=6.3 Hz,2H).

Compound 18 can be used for a bio-conjugation method according to theinvention.

N-((1-(4-(cyanoethynyl)phenyl)-1H-1,2,3-triazol-4-yl)methyl)-5-(dimethylamino)naphthalene-1-sulfonamide(20)

5-(dimethylamino)-N-(prop-2-yn-1-yl)naphthalene-1-sulfonamide (1 eq.,395 mg, 1.37 mmol) and 3-(4-azidophenyl)prop-2-ynenitrile (1 eq., 230mg, 1.37 mmol) were solubilized in tBuOH (6.91 mL). To this mixture wasadded a solution of copper sulfate pentahydrate (10%, 34.2 mg, 0.137mmol) in 0.5 mL of water followed by the solution of sodium ascorbate(0.5 eq., 135 mg, 0.685 mmol) in 0.5 mL of water. The resulting solutionwas stirred for 2 h and then concentrated on rotary evaporator. Theresidue was extracted with DCM. The organic layer was washed withsaturated aqueous solution of NH₄Cl and with water, dried over MgSO₄ andevaporated to give 20 (544 mg, 1.19 mmol, 87% yield) as a green solid.

¹H NMR (400 MHz, DMSO-d₆) δ=8.59 (br. s., 1H), 8.34 (d, J=7.3 Hz, 1H),8.33 (s, 1H) 8.26 (d, J=8.8 Hz, 1H), 8.13 (d, J=7.3 Hz, 1H), 8.00 (d,J=8.8 Hz, 2H), 7.83 (d, J=8.8 Hz, 2H), 7.61-7.51 (m, 2H), 7.18 (d, J=7.5Hz, 1H), 4.21 (s, 2H), 2.71 (s, 6H). ¹³C NMR (101 MHz, DMSO-d₆) δ=151.2,144.8, 138.4, 135.7, 135.6, 129.4, 128.9, 128.8, 128.6, 127.8, 123.4,121.3, 119.9, 119.0, 116.0, 114.9, 105.3, 82.5, 63.1, 44.9, 37.6.

Compound 20 can be used for a detection method (with dyes, for instance)according to the invention.

3-(4-isothiocyanatophenyl)propiolonitrile (21)

In a 50 mL RB flask, a solution of sodium hydrogen carbonate (886 mg,10.55 mmol) in 10 mL water was stirred for 10 min and to itdichloromethane (10 mL) was added followed by3-(4-aminophenyl)prop-2-ynenitrile (500 mg, 3.52 mmol). The reactionmixture was cooled to 0° C., thiophosgene (402 μL, 5.28 mmol) wasintroduced dropwise over a period of 30 min and continuously stirred atroom temperature for 1 h. The organic phase was separated and dried overanhydrous MgSO₄. Concentration of the solution afforded pure 21 (609 mg,3.31 mmol, 94% yield) in form of yellow solid.

¹H NMR (400 MHz, ACETONITRILE-d₃) δ=7.71 (d, J=8.5 Hz, 2H), 7.37 (d,J=8.5 Hz, 2H).

Compound 21 can be used for a bio-conjugation method according to theinvention.

tert-butyl(1-((4-(cyanoethynyl)phenyl)amino)-1-thioxo-6,9,12-trioxa-2-azapentadecan-15-yl)carbamate(22)

tert-Butyl (3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)carbamate (1eq., 91.5 mg, 0.271 mmol) was dissolved in of DCM (2 mL) and cooled to0° C. To this solution 3-(4-isothiocyanatophenyl)prop-2-ynenitrile (1eq., 50 mg, 0.271 mmol) in 1 mL of DCM was slowly added and the mixturewas stirred for 30 min. The reaction mix was concentrated to 1 mL andthe residue was purified by flash chromatography (DCM/MeOH gradient,100/0 to 90/10) to givetert-butyl(1-((4-(cyanoethynyl)phenyl)amino)-1-thioxo-6,9,12-trioxa-2-azapentadecan-15-yl)carbamate(126 mg, 0.25 mmol, 92% yield) as a yellow oil.

¹H NMR (400 MHz, METHANOL-d₄) δ=7.66 (s, 4H), 3.65-3.54 (m, 12H), 3.50(t, J=6.1 Hz, 2H), 3.12 (t, J=6.8 Hz, 2H), 1.91 (quin, J=6.1 Hz, 2H),1.72 (quin, J=6.4 Hz, 2H), 1.45 (s, 9H).

¹³C NMR (101 MHz, METHANOL-d₄) δ=182.0, 158.5, 144.6, 135.6, 106.4,84.8, 80.0, 71.6, 71.5, 71.3, 71.3, 70.0, 68.2, 63.0, 38.8, 31.0, 29.8,29.0

4-(cyanoethynyl)benzoyl chloride (23)

4-(2-cyanoeth-1-yn-1-yl)benzoic acid (1 eq., 30 mg, 0.175 mmol) wasdissolved in DCM (2 mL) and SOCl₂ (31.5 eq., 400 μL, 5.51 mmol) wasadded. The mixture was stirred at reflux until the solid completelydissolved and then evaporated to give pure 23 (29.6 mg, 0.156 mmol, 89%yield) as a white solid.

¹H NMR (400 MHz, CHLOROFORM-d) δ=8.08 (d, J=8.3 Hz, 2H), 7.67 (d, J=8.3Hz, 2H).

Compound 23 can be used for a bio-conjugation method according to theinvention.

tert-butyl(1-(4-(cyanoethynyl)phenyl)-1-oxo-6,9,12-trioxa-2-azapentadecan-15-yl)carbamate(24)

tert-Butyl(3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)carbamate (1eq., 50 mg, 0.156 mmol) and NEt₃ (5 eq., 78.9 mg, 0.108 mL, 0.78 mmol)were dissolved in 1 mL of DCM and cooled to −78° C. To this solution wasslowly added 23 (1 eq., 29.6 mg, 0.156 mmol) in 1 mL of DCM. The mixturewas gradually warmed to r.t. and stirred for 2 h. The reaction mix wasthen injected into flash chromatography column and eluted with DCM/MeOH(gradient 100/0 to 90/10) to give pure 24 (40.6 mg, 0.086 mmol, 55%) asa yellow oil.

¹H NMR (400 MHz, METHANOL-d₄) δ=7.92 (d, J=8.3 Hz, 2H), 7.82 (d, J=8.3Hz, 2H), 3.72-3.45 (m, 14H), 3.12 (t, J=6.8 Hz, 2H), 1.90 (quin, J=6.3Hz, 2H), 1.72 (quin, J=6.4 Hz, 2H), 1.44 (s, 9H).

4-(cyanoethynyl)-N-(15-oxo-4,7,10-trioxa-14-azanonatriaconta-24,26-diyn-1-yl)benzamide(25)

4-(2-cyanoeth-1-yn-1-yl)benzoic acid (1 eq., 29.7 mg, 0.173 mmol) wassuspended in DCM and SOCl₂ (39.8 eq., 820 mg, 0.5 mL, 6.89 mmol) wasadded. The mixture was stirred at reflux for 1.5 h, evaporated,dissolved in DCM, and added to the solution ofN-(3-{2-[2-(3-aminopropoxy)ethoxy]ethoxy}propyl)pentacosa-10,12-diynamide(1 eq., 100 mg, 0.173 mmol) and TEA (4 eq., 70.2 mg, 0.0964 mL, 0.693mmol) in DCM at −78° C. The resulting mixture was stirred at r.t. for 1h and evaporated. The residue was purified by flash chromatography(DCM/MeOH: 10/0 to 9/1) to give the desired product (35.4 mg, 0.0485mmol, 28% yield) as a yellow oil.

¹H NMR (400 MHz, CHLOROFORM-d) δ=7.90 (d, J=8.3 Hz, 2H), 7.67 (d, J=8.3Hz, 2H), 7.48 (br. s, 1H), 6.18 (br. s, 1H), 3.71-3.45 (m, 14H), 3.32(t, J=6.0 Hz, 2H), 2.23 (t, J=6.8 Hz, 4H), 2.15 (t, J=7.5 Hz, 2H), 1.90(td, J=6.0, 11.7 Hz, 2H), 1.73 (quin, J=6.1 Hz, 2H), 1.65-1.42 (m, 6H),1.41-1.32 (m, 4H), 1.32-1.19 (m, 22H), 0.88 (t, J=6.8 Hz, 3H).

Compound 25 can be used for the labeling (such as photolabeling) methodaccording to the invention or for binding and/or immobilizing compounds.

Ethyl(4-(cyanoethynyl)phenyl)carbamate (26)

To a solution of triphosgene (1 eq., 49.5 mg, 27.8 μL, 0.167 mmol) inDCM (4 mL) was added a solution of 3-(4-aminophenyl)prop-2-ynenitrile (3eq., 71.1 mg, 0.5 mmol) in DCM (1 mL). Then triethylamine (6 eq., 101mg, 138 μL, 1 mmol) in 1 mL of DCM was added dropwise. The mixture wasstirred for 15 min allowing the formation of isocyanate intermediate andthen ethanol (0.1 mL) was added dropwise. The reaction mixture wasstirred for 1 h then washed with 2×5 mL of water and evaporated. Theresidue was purified by flash chromatography to giveethyl(4-(cyanoethynyl)phenyl)carbamate (102 mg, 0.48 mmol, 96% yield) asa white solid.

¹H NMR (400 MHz, CHLOROFORM-d) δ=7.56 (d, J=8.5 Hz, 2H), 7.46 (d, J=8.5Hz, 2H), 6.79 (br. s., 1H), 4.26 (q, J=7.0 Hz, 2H), 1.33 (t, J=7.0 Hz,3H).

1-(4-(cyanoethynyl)phenyl)-3-(prop-2-yn-1-yl)urea (27)

To a solution of triphosgene (1 eq., 49.5 mg, 27.8 μL, 0.167 mmol) inDCM (2 mL) was added a solution of 3-(4-aminophenyl)prop-2-ynenitrile (3eq., 71.1 mg, 0.5 mmol) in DCM (3 mL). Then triethylamine (6 eq., 101mg, 138 μL, 1 mmol) was added, the mixture was stirred for 5 min andthen were added propargylamine (4.69 eq., 43 mg, 50.1 μL, 0.782 mmol)and triethylamine (2 eq., 33.7 mg, 46.3 μL, 0.333 mmol) in 1 mL of DCM.The reaction mixture was stirred for 1 h then washed with 5 mL of water,dried over MgSO₄ and concentrated. The residue was purified by flashchromatography (DCM/MeOH gradient) to give1-(4-(cyanoethynyl)phenyl)-3-(prop-2-yn-1-yl)urea (94.9 mg, 0.425 mmol,85% yield) as a white solid.

¹H NMR (400 MHz, METHANOL-d₄) δ=7.60 (d, J=8.8 Hz, 2H), 7.52 (d, J=8.8Hz, 2H), 4.00 (d, J=2.4 Hz, 2H), 2.61 (t, J=2.4 Hz, 1H).

Compound 27 can be used for click chemistry according to the invention.

prop-2-yn-1-yl(4-(cyanoethynyl)phenyl)carbamate (28)

To a solution of triphosgene (1 eq., 49.5 mg, 27.8 μL, 0.167 mmol) inDCM (2 mL) was added a solution of 3-(4-aminophenyl)prop-2-ynenitrile (3eq., 71.1 mg, 0.5 mmol) in DCM (3 mL). Then triethylamine (6 eq., 101mg, 138 μL, 1 mmol) was added. The mixture was stirred for 5 min andthen were added 2-propyn-1-ol (6 eq., 56.1 mg, 59.1 μL, 1 mmol) andtriethylamine (2 eq., 33.7 mg, 46.3 μL, 0.333 mmol) in 1 mL of DCM. Thereaction mixture was stirred for 1 h then washed with 5 mL of water,dried over MgSO₄ and concentrated. The residue was purified by flashchromatography (Cyclohexane/EtOAc gradient) to give prop-2-yn-1-yl(4-(cyanoethynyl)phenyl)carbamate (104 mg, 0.465 mmol, 93% yield) as awhite solid.

¹H NMR (400 MHz, CHLOROFORM-d) δ=7.57 (d, J=8.6 Hz, 2H), 7.48 (d, J=8.6Hz, 2H), 4.80 (d, J=2.3 Hz, 2H), 2.54 (t, J=2.3 Hz, 1H).

Compound 28 can be used for click chemistry according to the invention.

bicyclo[6.1.0]non-4-yn-9-ylmethyl(4-(cyanoethynyl)phenyl)carbamate (29)

To a solution of triphosgene (1 eq., 34.8 mg, 19.5 μL, 0.117 mmol) inDCM (4 mL) was added a solution of 3-(4-aminophenyl)prop-2-ynenitrile (3eq., 50 mg, 0.352 mmol) in DCM (1 mL). Then triethylamine (6 eq., 71.2mg, 97.8 μL, 0.703 mmol) was added dropwise. The mixture was stirred for5 min at r.t. and then bicyclo[6.1.0]non-4-yn-9-ylmethanol (3 eq., 52.8mg, 0.352 mmol) and triethylamine (2 eq., 23.7 mg, 32.6 μL, 0.234 mmol)were added in 1 mL of DCM. The reaction mixture was stirred at r.t. for2 hours. After full conversion was confirmed by HPLC the mixture wasconcentrated to 1 mL volume and purified by flash chromatography(cyclohexane/EtOAc gradient) to givebicyclo[6.1.0]non-4-yn-9-ylmethyl(4-(cyanoethynyl)phenyl)carbamate (68.3mg, 0.215 mmol, 183%) as a white solid.

¹H NMR (400 MHz, CHLOROFORM-d) δ=7.55 (d, J=8.8 Hz, 2H), 7.47 (d, J=8.8Hz, 2H), 7.09 (br. s, 1H), 2.38-2.14 (m, 6H), 1.67-1.51 (m, 2H), 1.42(quin, J=8.7 Hz, 1H), 1.04-0.91 (m, 2H)

Compound 29 can be used for click chemistry (such as strain-promotedclick) according to the invention.

3-(4-((trimethylsilyl)ethynyl)phenyl)prop-2-yn-1-ol (30a)

Product was synthesized according to general procedure B for coupling.Yield: 99%.

¹H NMR (400 MHz, CHLOROFORM-d) δ=7.41 (d, J=8.4 Hz, 2H), 7.36 (d, J=8.4Hz, 2H), 4.50 (d, J=5.5 Hz, 2H), 1.89 (t, J=5.5 Hz, 1H), 0.25 (s, 9H).

3-(4-((trimethylsilyl)ethynyl)phenyl)propiolonitrile (30)

Product was synthesized according to general procedure ofMnO₂-oxidation. Reaction time: 3 hours; yield: 29%.

¹H NMR (400 MHz, CHLOROFORM-d) δ=7.55 (d, J=8.4 Hz, 2H), 7.48 (d, J=8.4Hz, 2H), 0.27 (s, 9H).

¹³C NMR (101 MHz, CHLOROFORM-d) δ=133.2, 132.2, 126.9, 117.1, 105.3,103.4, 99.4, 82.3, 64.5, −0.3.

3,3′-(5-amino-1,3-phenylene)dipropiolonitrile (31)

Product was synthesized according to general procedure ofMnO₂-oxidation. Reaction time: 3 hours; yield: 11%.

¹H NMR (400 MHz, CHLOROFORM-d) δ=7.20 (t, J=1.3 Hz, 1H), 6.98 (d, J=1.3Hz, 2H), 4.02 (br. s., 2H).

¹³C NMR (101 MHz, CHLOROFORM-d) δ=147.1, 127.5, 121.6, 119.5, 105.0,81.0, 63.6.

Compound 31 can be used for rebridging (diAPN) according to theinvention.

3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)propiolonitrile(32)

Product was synthesized according to general procedure ofMnO₂-oxidation. Reaction time: 4 hours; yield: 63%.

¹H NMR (400 MHz, CHLOROFORM-d) δ=7.84 (d, J=8.2 Hz, 2H), 7.60 (d, J=8.2Hz, 2H), 1.36 (s, 12H).

3,3′-(1,2-Phenylene)dipropiolonitrile (33)

33a: 3,3′-(1,2-Phenylene)bis(prop-2-yn-1-ol)

To the degassed solution of 1,2-diiodobenzene (1 eq., 661 mg, 0.262 mL,2 mmol) and propargylic alcohol (2.3 eq., 272 μL, 4.61 mmol) in butylamine (15.8 mL), Pd(PPh₃)₄ (4%, 92.6 mg, 0.0801 mmol) was added and theobtained reaction mass was refluxed overnight. Solvents were evaporatedand the obtained crude product was purified by flash chromatography (20minutes gradient EtOAc/Cyclohexane) to yield 33a (150 mg, 0.8 mmol, 40%)as a brownish solid.

¹H NMR (400 MHz, METHANOL-d₄) δ 7.38-7.53 (m, 2H), 7.25-7.38 (m, 2H),4.48 (s, 4H); ¹³C NMR (101 MHz, METHANOL-d₄) d 135.6, 131.9, 129.2,95.6, 86.6, 53.9; ESI-MS: C₁₂H₁₁O₂ ⁺ [M+H]⁺, 187.1; found 187.1.

33: 3,3′-(1,2-Phenylene)dipropiolonitrile

The compound was obtained as the only product of the standard MnO₂oxidation protocol. Reaction time: 75 minutes. Brown solid, yield: 42%.

¹H NMR (400 MHz, METHANOL-d₄) δ 7.89 (dd, J=3.30, 5.80 Hz, 2H), 7.73(dd, J=3.30, 5.80 Hz, 2H); ¹³C NMR (101 MHz, METHANOL-d₄) δ 136.0,133.5, 126.5, 105.5, 80.2, 67.2; GC-ESI-MS: C₁₂H₅N₂ ⁻ [M+H]⁻, 177.0;found 177.0.

3,3′-(1,3-Phenylene)dipropiolonitrile (34)

34a: 3,3′-(1,3-Phenylene)bis(prop-2-yn-1-ol)

Same procedure as for the synthesis of 33a. Brownish solid, yield: 55%.

¹H NMR (400 MHz, METHANOL-d₄) δ 7.47 (s, 1H), 7.36-7.43 (m, 2H),7.29-7.36 (m, 1H), 4.41 (s, 4H); ¹³C NMR (101 MHz, METHANOL-d₄) δ 135.3,132.5, 129.8, 124.8, 89.7, 84.5, 51.2; ESI-MS: C₁₂H₁₁O₂ ⁺ [M+H]⁺, 187.1;found 187.0.

34: 3,3′-(1,3-Phenylene)dipropiolonitrile

The compound was obtained as the only product of the standard MnO₂oxidation protocol. Reaction time: 2 hours. Brown solid, yield: 35%.

¹H NMR (400 MHz, METHANOL-d₄) δ 8.10 (d, J=1.50 Hz, 1H), 7.93 (dd,J=1.50, 8.00 Hz, 1H), 7.63 (t, J=8.00 Hz, 2H); ¹³C NMR (101 MHz,METHANOL-d₄) δ 139.3, 137.8, 131.2, 120.0, 105.7, 81.7, 64.2; GC-ESI-MS:C₁₂H₅N₂ ⁺ [M+H]⁺, 177.0; found 177.1.

3,3′-(1,4-Phenylene)dipropiolonitrile (35)

35a: 3,3′-(1,4-Phenylene)bis(prop-2-yn-1-ol)

Same procedure as for the synthesis of 33a, but refluxed for 72 hours.Brownish solid, yield: 35%.

¹H NMR (400 MHz, METHANOL-d₄) δ 7.39 (s, 4H), 4.41 (s, 4H); ¹³C NMR (101MHz, METHANOL-d₄) δ 132.6, 124.3, 101.4, 90.8, 84.9, 51.2; ESI-MS:C₁₂H₁₁O₂ ⁺ [M+H]⁺, 187.1; found 187.1.

35: 3,3′-(1,4-Phenylene)dipropiolonitrile

The compound was obtained as the only product of the standard MnO₂oxidation protocol. Reaction time: 2 hours. Brown solid, yield: 19%.

¹H NMR (400 MHz, METHANOL-d₄) δ7.94 (s, 4H); ¹³C NMR (101 MHz,METHANOL-d₄) δ135.0, 121.6, 105.5, 82.0, 65.9; GC-ESI-MS: C₁₂H₅N₂ ⁺[M+H]⁺, 177.0; found 177.0.

Compounds 33-35 can be used for rebridging (diAPN) according to theinvention.

tert-butyl(S)-2-((tert-butoxycarbonyl)amino)-3-(4-(((trifluoromethyl)sulfonyl)oxy)phenyl)propanoate(36b)

To a cooled to 0° C. solution of tert-butyl2-{[(tert-butoxy)carbonyl]amino}-3-(4-hydroxyphenyl)propanoate (1 eq.,518 mg, 1.54 mmol) in pyridine (2.5 mL), triflic anhydride (1.1 eq., 476mg, 0.28 mL, 1.69 mmol) was added dropwise over 20 minutes (usingsyringe presser). The resulting dark solution was let to warm up to roomtemperature, poured into water (10 mL), and extracted with ethyl ester(15 mL). The ether extract was washed sequentially with water (5 mL), 1NHCl (2×5 mL), water (5 mL), brine (5 mL), dried over MgSO₄, andevaporated to give the targeted product (614 mg, 1.31 mmol, 85%) as adark-red oil. The product was used in the next step without furtherpurification.

¹H NMR (400 MHz, CHLOROFORM-d) δ 7.03-7.18 (m, 3H), 6.82-7.03 (m, 2H),4.87 (d, J=7.28 Hz, 1H), 4.24 (d, J=7.03 Hz, 1H), 2.65-2.95 (m, 2H),1.17 (s, 9H), 1.21 (s, 9H).

¹³C NMR (101 MHz, CHLOROFORM-d) δ 170.5, 148.5, 137.3, 131.3, 121.1,120.3, 117.1, 82.5, 80.0, 54.7, 38.1, 28.3, 27.9.

tert-butyl(S)-2-((tert-butoxycarbonyl)amino)-3-(4-(3-hydroxyprop-1-yn-1-yl)phenyl)propanoate(36a)

To a solution of phenoltryphlate (1 eq., 136 mg, 0.291 mmol) inmorpholine (1 mL) were consequently added PdCl₂(PPh₃)₂ (5%, 10.2 mg,0.0145 mmol), CuI (10%, 5.53 mg, 0.0291 mmol), and propargylic alcohol(2 eq., 32.6 mg, 0.0343 mL, 0.581 mmol). The obtained reaction mixturewas degassed and heated at 60° C. for 24 hours. The resulting blacksolution was poured into water (10 mL), extracted with EtOAc (3×10 mL).The united organic layers were washed with 1N HCl (2×10 mL), water (1×10mL), dried over MgSO₄ and evaporated to give crude product, which afterpurification by flash chromatography gave the targeted product (8.73 mg,0.0232 mmol, 8%) as a yellowish solid.

¹H NMR (400 MHz, METHANOL-d₄) δ 7.30-7.45 (m, J=7.78 Hz, 2H), 7.14-7.30(m, J=8.03 Hz, 2H), 4.40 (s, 2H), 4.18-4.32 (m, 1H), 3.06 (dd, J=6.27,13.80 Hz, 1H), 2.91 (dd, J=8.66, 13.68 Hz, 1H), 1.45-1.53 (m, 1H), 1.42(d, J=3.26 Hz, 19H).

tert-Butyl2-((tert-butoxycarbonyl)amino)-3-(4-(cyanoethynyl)phenyl)propanoate (36)

Product was synthesized according to general procedure ofMnO₂-oxidation. Reaction time: 2 hours; yield: 56%.

¹H NMR (400 MHz, CHLOROFORM-d) δ=7.54 (d, J=8.2 Hz, 2H), 7.24 (d, J=8.2Hz, 2H), 5.05 (d, J=7.3 Hz, 1H), 4.46 (td, J=6.1, 7.3 Hz, 1H), 3.14 (dd,J=6.1, 13.7 Hz, 1H), 3.05 (dd, J=6.1, 13.7 Hz, 1H), 1.42 (s, 9H), 1.41(s, 9H).

¹³C NMR (101 MHz, CHLOROFORM-d) δ=170.3, 154.9, 141.4, 133.4, 130.1,115.9, 105.5, 82.9, 82.5, 79.9, 63.2, 54.5, 38.8, 28.3, 27.9.

Compound 36 can be used for purification and/or immobilization accordingto the invention.

4-(cyanoethynyl)-N-(2-(2-(2-(5-(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)ethoxy)ethoxy)ethyl)benzamide(37)

To the solution ofN-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide(1 eq., 222 mg, 0.593 mmol) in dry DMF (1 mL) was added sodium4-((4-(cyanoethynyl)benzoyl)oxy)-2,3,5,6-tetrafluorobenzenesulfonate(1.2 eq., 300 mg, 0.712 mmol) and DIEA (5.1 eq., 391 mg, 0.5 mL, 3.03mmol). The mixture was stirred at r.t. for 3 hours and then purified bysemi-preparative HPLC to give the desired product (68.8 mg, 0.13 mmol,22% yield) as a yellow oil.

¹H NMR (400 MHz, METHANOL-d₄) δ=7.92 (d, J=8.5 Hz, 2H), 7.81 (d, J=8.5Hz, 2H), 4.49 (dd, J=4.8, 7.8 Hz, 1H), 4.30 (dd, J=4.5, 7.8 Hz, 1H),3.71-3.56 (m, 8H), 3.54 (t, J=5.5 Hz, 2H), 3.34 (t, J=5.5 Hz, 2H),3.24-3.14 (m, 1H), 2.92 (dd, J=4.8, 12.8 Hz, 1H), 2.70 (d, J=12.8 Hz,1H), 2.19 (t, J=7.4 Hz, 2H), 1.78-1.50 (m, 4H), 1.48-1.35 (m, 2H).

¹³C NMR (101 MHz, METHANOL-d₄) δ=176.3, 168.8, 166.2, 138.9, 135.0,129.1, 121.5, 105.9, 83.1, 71.5, 71.4, 70.7, 70.6, 64.6, 63.5, 61.8,57.1, 41.2, 40.4, 36.9, 29.9, 29.6, 27.0.

2-((4-((4-(Cyanoethynyl)phenyl)amino)-4-oxobutyl)-amino)-2-oxoethyl)tris(2,4,6-trimethoxyphenyl)phosphoniumtrifluoroacetate (38)

38d: tert-Butyl(4-((4-iodophenyl)amino)-4-oxobutyl)carbamate

To the cooled to 0° C. solution of Boc-GABA (1 eq., 0.928 g, 4.57 mmol),TEA (3 eq., 1.39 g, 1.9 mL, 13.7 mmol) and DMAP (0.05 eq., 0.0279 g,0.228 mmol) in DCM (11.7 mL), EDC (1 eq., 0.875 g, 4.57 mmol) was added.The obtained reaction mass was stirred for another 10 minutes at 0° C.,an ice bath was removed, and p-iodoaniline (1 eq., 1 g, 4.57 mmol) wasadded and the reaction was left overnight at 25° C. The obtainedreaction mass was washed with 1M HCl (2×20 mL), water (1×20 mL), anddried over Na₂SO₄ to give 38d (1125 mg, 2.79 mmol, 61%), which was usedwithout further purification.

¹H NMR (400 MHz, CHLOROFORM-d) δ 9.04 (br. s., 1H), 7.58-7.72 (m, J=8.50Hz, 2H), 7.37-7.51 (m, J=8.50 Hz, 2H), 4.81 (br. s., 1H), 3.27 (m, 2H),2.30-2.50 (m, 2H), 1.88 (m, 2H), 1.49 (s, 9H); ¹³C NMR (101 MHz,CHLOROFORM-d) δ 174.2, 157.4, 137.8, 120.9, 120.0, 87.3, 77.0, 33.1,32.8, 28.4, 26.0; ESI-MS: C₁₅H₂₂N₂O₃ ⁺ [M+H]⁺, 405.0; found 405.1.

38c:tert-Butyl(4-((4-(3-hydroxyprop-1-yn-1-yl)phenyl)-amino)-4-oxobutyl)carbamate

Synthesised following the protocol B for Sonogashira coupling. Yellowishsolid, yield: 79%.

¹H NMR (400 MHz, METHANOL-d₄) δ 7.54-7.58 (m, J=8.50 Hz, 2H), 7.34-7.38(m, J=8.50 Hz, 2H), 4.40 (s, 2H), 3.13 (t, J=6.90 Hz, 2H), 2.41 (t,J=7.40 Hz, 2H), 1.81-1.89 (m, 2H), 1.44 (s, 9H); ¹³C NMR (101 MHz,METHANOL-d₄) δ 174.0, 158.6, 140.1, 133.2, 120.8, 119.5, 88.3, 85.3,80.1, 51.3, 40.9, 35.3, 28.8, 27.1; ESI-MS:

C₁₈H₂₅N₂O₄ ⁺ [M+H]⁺, 332.1; found 332.0.

38b: tert-Butyl(4-((4-(cyanoethynyl)phenyl)amino)-4-oxobutyl)carbamate

Synthesised using standard protocol of MnO₂ oxidation. Reaction time: 1hour. White solid, yield: 85%.

¹H NMR (400 MHz, METHANOL-d₄) δ 7.61-7.65 (m, J=8.80 Hz, 2H), 7.54-7.59(m, J=8.50 Hz, 2H), 3.04 (t, J=6.85 Hz, 2H), 2.34 (t, J=7.40 Hz, 2H),1.74-1.81 (m, 2H), 1.34 (s, 9H); ¹³C NMR (101 MHz, METHANOL-d₄) δ 174.3,159.1, 143.8, 135.7, 120.8, 112.8, 106.3, 84.6, 62.7, 40.8, 35.3, 34.8,28.8, 26.9; ESI-MS: C₁₈H₂₂N₃O₃ ⁺ [M+H]⁺, 328.1; found 328.1.

38a: 4-((4-(Cyanoethynyl)phenyl)amino)-4-oxobutan-1-aminiumtrifluoroacetate

To a suspension of 38b (1 eq., 62.8 mg, 0.192 mmol) in DCM (1 mL), TFA(20 eq., 285 μL, 3.83 mmol) was added and the obtained solution wasstirred at 25° C. for 30 minutes. The target product 38a (TFA salt, 65.0mg, 0.19 mmol, 99%) was obtained after the evaporation of the reactionmass and was used without further purification in the next step.

¹H NMR (400 MHz, METHANOL-d₄) δ 7.71-7.79 (m, J=9.15 Hz, 2H), 7.63-7.70(m, J=9.15 Hz, 2H), 3.04 (t, J=6.80 Hz, 2H), 2.6 (t, J=7.05 Hz, 2H),1.98-2.08 (m, 2H); ¹³C NMR (101 MHz, METHANOL-d₄) δ 173.1, 143.6, 135.7,120.7, 112.9, 106.2, 84.5, 62.7, 40.4, 34.5, 24.0; ESI-MS: C₁₃H₁₄N₃₀ ⁺[M+H]⁺, 228.1; found 228.1.

38:(2-((4-((4-(Cyanoethynyl)phenyl)amino)-4-oxobutyl)amino)-2-oxoethyl)tris(2,4,6-trimethoxyphenyl)phosphoniumtrifluoroacetate

To the solution of 38a (1 eq., 10.1 mg, 0.0296 mmol) in DMF (250 μL),TEA (1 eq., 4 μL, 0.0296 mmol) was added. TMPP-Ac-OSu (1 eq., 22.7 mg,0.0296 mmol) was added to the obtained solution and the reaction masswas for 15 minutes at room temperature.

The crude product was purified by HPLC to isolate 38 (9.9 mg, 0.0126mmol, 42%) as a main product.

¹H NMR (400 MHz, METHANOL-d₄) δ 7.44-7.59 (m, 4H), 6.13 (d, J=4.52 Hz,6H), 3.75 (s, 9H), 3.50 (s, 18H), 3.00 (td, J=7.91, 15.31 Hz, 2H), 2.26(t, J=6.90 Hz, 2H), 1.64-1.75 (m, 2H), 1.25-1.43 (m, 2H); ¹³C NMR (101MHz, METHANOL-d₄) δ 174.2, 167.4, 167.4, 165.3, 143.6, 135.8, 120.6,112.8, 106.3, 92.2 (d, J=8 Hz), 84.5, 62.7, 56.5, 56.2, 37.5, 29.4 (d,J=64 Hz), 27.9, 27.7, 24.9; ESI-HRMS: C₄₂H₄₇N₃O₁₁P⁺ [M]⁺, 800.29427;found 800.29401.

(5-((4-(Cyanoethynyl)phenyl)amino)-5-oxopentyl)tris(2,4,6-trimethoxyphenyl)phosphoniumbromide (39)

39d: 5-Bromopentanoyl chloride

Degassed solution of 5-bromopentanoic acid (1 eq., 2.85 g, 15.7 mmol)and SOCl₂ (1 eq., 1.87 g, 1.14 mL, 15.7 mmol) in DCM (50 mL) wasrefluxed for 3 hours. The obtained reaction mass was evaporated underreduced pressure to give 39d (3.11 g, 100%) as a yellowish oil. Thecrude product was used in the next step without purification.

39c: 5-Bromo-N-(4-iodophenyl)pentanamide

Solution of 39d (1 eq., 3.11 g, 15.7 mmol) in DCM (50 mL) was pouredinto a cooled to −78° C. solution of 4-iodoaniline (1 eq., 3.45 g, 15.7mmol) and DIPEA (1 eq., 2.03 g, 2.6 mL, 15.7 mmol) in DCM (50 mL).Obtained reaction mass was allowed to warm to room temperature, stirredfor another 30 min, washed with 1N HCl (2×25 mL), water (1×25 mL), driedover Na₂SO₄ and evaporated to give 39c (5.60 g, 14.66 mmol, 93%) asbrown solid.

¹H NMR (400 MHz, METHANOL-d₄) δ 7.62-7.66 (m, 2H), 7.38-7.43 (m, 2H),3.50 (t, J=6.53 Hz, 2H), 2.42 (t, J=7.28 Hz, 2H), 1.81-1.98 (m, 4H),1.37-1.42 (m, 1H); ¹³C NMR (101 MHz, METHANOL-d₄) δ 174.0, 140.5, 138.9,123.1, 87.6, 36.9, 33.8, 33.4, 25.3; ESI-MS: C₁₁H₁₄BrINO⁺ [M+H]⁺, 381.9;found 381.8.

39b: 5-Bromo-N-(4-(3-hydroxyprop-1-yn-1-yl)phenyl)pentanamide

Synthesised following the protocol A for Sonogashira coupling. Brownsolid, yield: 92%.

¹H NMR (400 MHz, METHANOL-d₄) δ 7.49-7.63 (m, J=8.53 Hz, 2H), 7.32-7.43(m, J=8.53 Hz, 2H), 4.40 (s, 2H), 4.26 (s, 1H), 3.50 (t, J=6.53 Hz, 2H),2.43 (t, J=7.15 Hz, 2H), 1.90-2.04 (m, 2H), 1.74-1.90 (m, 2H), 1.32 (s,1H); ¹³C NMR (101 MHz, METHANOL-d₄) δ 173.9, 140.0, 133.1, 120.7, 119.6,88.2, 85.2, 51.2, 36.8, 33.7, 33.3, 25.3; ESI-MS: C₁₄H₁₇BrNO⁺ [M+H]⁺,310.0; found 310.0.

39a: 5-Bromo-N-(4-(cyanoethynyl)phenyl)pentanamide

2M solution of NH₃ (4 eq., 94.8 mg, 5.56 mmol) in IPA and anhydrousMgSO₄ (15 eq., 2511 mg, 20.9 mmol) were added to a stirred solution of39b (1 eq., 431 mg, 1.39 mmol) in THF (3.42 mL). Activated MnO₂ (15 eq.,1814 mg, 20.9 mmol) was added to the solution and the resulting mixturewas stirred at room temperature for 4 hours (controlled by TLC, no morestarting alcohol; NB: too long reaction time gives hydrolysis product),diluted with DCM (13 mL). The mixture was filtered, washed thoroughlywith DCM and the combined filtrates were concentrated under reducedpressure. The solid residue was purified by flash chromatography(EtOAc-cyclohexane, 20 min gradient from 0 to 100% of EtOAc) to give 39as a white solid (288 mg, 0.946 mmol, 68%).

¹H NMR (400 MHz, METHANOL-d₄) δ 7.69-7.79 (m, J=8.78 Hz, 2H), 7.59-7.69(m, J=8.78 Hz, 2H), 3.50 (t, J=6.53 Hz, 2H), 1.79-1.99 (m, 4H), 1.26 (t,J=7.15 Hz, 2H); ¹³C NMR (101 MHz, METHANOL-d₄) δ 174.3, 143.8, 135.7,120.7, 112.8, 106.2, 101.4, 84.6, 37.0, 33.7, 33.4, 25.2. ESI-MS:C₁₄H₁₄BrN₂O⁺ [M+H]⁺, 304.0; found 304.0.

39:(5-((4-(Cyanoethynyl)phenyl)amino)-5-oxopentyl)tris(2,4,6-trimethoxy-phenyl)phosphoniumbromide

39a (1 eq., 20 mg, 0.0655 mmol) andtris(2,4,6-trimethoxy-phenyl)phosphane (TMPP, 1.2 eq., 41.9 mg, 0.0786mmol) were dissolved in dry toluene (1 mL) and stirred overnight at roomtemperature. 39 (TFA salt, 22 mg, 39%) was obtained after reverse-phaseHPLC as a white solid.

¹H NMR (400 MHz, METHANOL-d₄) δ 7.51-7.55 (m, 4H), 6.13 (d, J=4.77 Hz,6H), 3.75 (s, 9H), 3.50 (s, 18H), 3.00 (td, J=6.90, 15.31 Hz, 2H), 2.26(t, J=6.90 Hz, 2H), 1.73 (m, 2H), 1.22-1.45 (m, 2H); ¹³C NMR (101 MHz,METHANOL-d₄) δ 174.2, 167.4, 165.3, 143.6, 135.8, 120.6, 112.8, 106.2,93.6, 92.3, 92.2, 84.5, 62.7, 56.3, 37.5, 29.7, 27.7, 24.9; ESI-HRMS:C₄₁H₄₆N₂O₁₀P⁺ [M]⁺, 757.28846; found 757.29552.

(4-(4-(Cyanoethynyl)benzamido)butyl)tris(2,4,6-trimethoxyphenyl)phosphoniumtrifluoroacetate (40)

40c: N-(4-Bromobutyl)-4-iodobenzamide

4-iodobenzoic acid (1 eq., 1.45 g, 5.85 mmol) was heated at 110° C. inSOCl₂ (9 eq., 3.8 mL, 52.6 mmol) until complete dissolving (around 15min). Excess of SOCl₂ was removed in vacuo and obtained solid was pouredinto DCM (15 mL), cooled to −78° C. and DIPEA (3.1 eq., 3 mL, 18.2 mmol)was added under vigorous stirring. 3-Bromopropylamine hydrobromide (1.5eq., 1.90 g, 8.77 mmol) was added to the obtained reaction mass was leftstirring for 5 minutes still at −78° C., let to warm up to roomtemperature, while stirring for another 20 minutes. Ethyl acetate (100mL) was added with 1M HCl (5 mL), obtained solid was filtered (product),washed with water and dried to yield 40c (2.09 g, 5.67 mmol, 97%) as awhite solid.

¹H NMR (400 MHz, METHANOL-d₄) δ 7.85 (m, J=8.40 Hz, 2H), 7.58 (m, J=8.40Hz, 2H), 3.48-3.56 (m, 4H), 2.12-2.23 (m, 2H); ¹³C NMR (101 MHz,METHANOL-d₄) δ 160.1, 139.2, 136.0, 132.8, 102.4, 50.1, 43.2, 23.0;ESI-MS: C₁₀H₁₂BrINO⁺ [M+H]⁺, 367.9; found 368.0.

40b: N-(4-Bromobutyl)-4-(3-hydroxyprop-1-yn-1-yl)benzamide

Synthesised following the protocol B for Sonogashira coupling. Brownsolid, yield: 81%.

¹H NMR (400 MHz, METHANOL-d₄) δ 7.73 (m, J=8.40 Hz, 2H), 7.38 (m, J=8.40Hz, 2H), 4.37 (t, J=5.30 Hz, 2H), 4.34 (s, 2H), 3.51 (t, J=5.80 Hz, 2H),1.92-1.98 (m, 2H); ¹³C NMR (101 MHz, METHANOL-d₄) δ 159.2, 134.2, 132.4,128.2, 127.1, 91.2, 84.8, 67.2, 51.2, 43.3, 22.5; ESI-MS: C₁₃H₁₅BrNO₂ ⁺[M+H]⁺, 295.0; found 295.0.

40a: N-(3-Bromopropyl)-4-(cyanoethynyl)benzamide

Synthesised using standard protocol of MnO₂ oxidation. Reaction time: 45minutes. Brown solid, yield: 52%.

¹H NMR (400 MHz, METHANOL-d₄) δ 7.90 (m, J=8.50 Hz, 2H), 7.80 (m, J=8.50Hz, 2H), 3.42-3.55 (m, 2H), 3.25-3.35 (m, 2H), 2.13-2.23 (m, 2H),1.92-1.98 (m, 2H); ¹³C NMR (101 MHz, METHANOL-d₄) δ 168.7, 138.8, 134.9,128.9, 121.4, 105.8, 83.0, 67.3, 41.9, 39.8, 22.8; ESI-MS: C₁₃H₁₂BrN₂O⁺[M+H]⁺, 291.0; found 291.2.

40:(4-(4-(Cyanoethynyl)benzamido)butyl)tris(2,4,6-trimethoxyphenyl)phosphoniumtrifluoroacetate

40a (1 eq., 30 mg, 0.103 mmol) and tris(2,4,6-trimethoxyphenyl)phosphane(TMPP, 1 eq., 54.9 mg, 0.103 mmol) were dissolved in dry toluene (2 mL).The obtained solution was left overnight at room temperature. Theprecipitate was filtered, resolubilised in DMSO, and purified by HPLC togive 40 (35 mg, 0.0409 mmol, 40%) as a white solid.

¹H NMR (400 MHz, METHANOL-d₄) δ 7.75 (d, J=8.50 Hz, 2H), 7.69 (d, J=8.50Hz, 2H), 6.16 (d, J=4.70 Hz, 2H), 3.76 (s, 9H), 3.51 (s, 18H), 3.35 (t,J=7.10 Hz, 2H), 2.98-3.10 (m, 2H), 1.53-1.64 (m, 2H); ¹³C NMR (101 MHz,METHANOL-d₄) δ 168.5, 167.5, 165.3, 138.7, 134.9, 128.8, 121.2, 105.8,94.0, 92.9, 92.3, 82.9, 64.5, 56.5, 41.7, 27.8, 25.7; ESI-HRMS:C₄₀H₄₄N₂O₁₀P⁺ [M]⁺, 743.22728; found 743.23946.

Compounds 38-40 can be used for detection and/or separation methodaccording to the invention.

3-(4-(2,5-Dioxo-2,5-dihydro-1H-1-pyrrol-1-yl)phenyl)propiolonitrile (41)

41a: (Z)-4-((4-(Cyanoethynyl)phenyl)amino)-4-oxobut-2-enoic acid

To the solution of 7 (1 eq., 76.8 mg, 0.541 mmol) in acetone (2 mL),maleic anhydride (2 eq., 106 mg, 1.08 mmol) was added. A yellowish solidwas obtained after about 7 hours of stirring. The reaction mass wasevaporated, an excess of maleic anhydride and maleic acid was washedwith methanol. 41a (127 mg, 0.53 mmol, 98%) was obtained as yellowishsolid, no further purification was needed.

¹H NMR (400 MHz, DMSO-d₆) δ 12.90 (br. s., 1H), 10.70 (s, 1H), 7.62-7.90(m, 4H), 6.50 (d, J=11.90 Hz, 1H), 6.34 (d, J=11.90 Hz, 1H); ¹³C NMR(101 MHz, DMSO-d₆) δ 166.8, 163.8, 142.4, 135.0, 131.7, 130.1, 119.3,110.2, 105.6, 84.3, 61.9; ESI-MS: C₁₃H₇N₂O₃ ⁻ [M−H]⁻, 239.0; found239.0.

41: 3-(4-(2,5-Dioxo-2,5-dihydro-1H-1-pyrrol-1-yl)phenyl)-propiolonitrile

To the solution of 41a (1 eq., 75 mg, 0.312 mmol) in dry DMF (1.21 mL)trifluoroacetic anhydride (2 eq., 86.9 μL, 0.624 mmol) was added.Stirring continued for another 5 minutes at room temperature and K₂CO₃(3 eq., 129 mg, 0.937 mmol) was added. The reaction mass stirred foranother 60 minutes, then directly purified by HPLC to give 41 (65.9 mg,0.297 mmol, 95%) as a slightly yellow solid.

¹H NMR (400 MHz, DMSO-d₆) δ 7.81 (m, J=8.50 Hz, 2H), 7.52 (m, J=8.50 Hz,2H), 6.96 (s, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 169.0, 134.4, 134.0,126.0, 117.3, 117.0, 82.2, 78.5, 62.3; ESI-MS: C₁₃H₇N₂O₂ ⁺ [M+H]⁺,223.0; found 229.9.

Compound 41 can be used for a bioconjugation method according to theinvention.

2,5-Dioxopyrrolidin-1-yl5-((4-(cyanoethynyl)-phenyl)-amino)-5-oxopentanoate (42)

42a: 5-((4-(Cyanoethynyl)phenyl)amino)-5-oxopentanoic acid

To a solution of 7 (1 eq., 200 mg, 1.41 mmol) in acetone (1 mL),glutaric anhydride (2 eq., 321 mg, 2.81 mmol) was added. The obtainedsolution was stirred for 24 hours at room temperature. Acetone wasevaporated, the crude product was recrystallised from IPA-cyclohexane togive 42a (324 mg, 1.27 mmol, 90%) as a grey solid.

¹H NMR (400 MHz, DMSO-d₆) δ 10.21 (s, 1H), 8.15 (br. s., 1H), 7.60 (d,J=8.72 Hz, 2H), 7.52 (d, J=8.72 Hz, 2H), 2.52-2.62 (m, 4H), 2.22-2.32(m, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ170.0, 168.5, 140.9, 134.2, 119.0,111.9, 105.4, 84.1, 63.3, 30.1, 29.0, 21.2; ESI-MS: C₁₄H₁₁N₂O₃ ⁻ [M−H]⁻,255.1; found 255.1.

42: 2,5-Dioxopyrrolidin-1-yl5-((4-(cyanoethynyl)phenyl)-amino)-5-oxopentanoate

To a solution of 42a (1 eq., 18 mg, 0.0702 mmol) in DCM (1 mL), DCC(1.02 eq., 14.8 mg, 0.0716 mmol) and TEA (1 eq., 6.52 mg, 0.00895 mL,0.0644 mmol) were added. The obtained reaction mass was stirred for 5minutes, NHS (1 eq., 8.08 mg, 0.0702 mmol) was added. The resultingsolution stirred for another 2 hours at room temperature. The crudeproduct was purified by flash chromatography (cyclohexane-EtOAc) to give42 (6.45 mg, 0.0183 mmol, 26%) as a white solid.

¹H NMR (400 MHz, CHLOROFORM-d) δ 8.27 (br. s., 1H), 7.64 (d, J=8.78 Hz,2H), 7.57 (d, J=8.78 Hz, 2H), 2.94 (s, 4H), 2.74 (t, J=6.53 Hz, 2H),2.52 (t, J=6.90 Hz, 2H), 2.23 (m, 2H); ¹³C NMR (101 MHz, CHLOROFORM-d) d170.3, 169.5, 168.2, 141.3, 134.6, 119.4, 112.4, 105.7, 83.2, 62.9,35.6, 29.9, 25.7, 21.2; ESI-MS: C₁₈H₁₆N₃O₅ ⁺ [M+H]⁺, 353.1; found 353.2.

Compound 42 can be used for a bioconjugation method according to theinvention.

3-(4-azidophenyl)propiolonitrile (43)

7 (1 eq., 151 mg, 1.07 mmol) was dissolved in acetonitrile (2.34 mL) ina 25 mL roundbottomed flask and cooled to 0° C. in an ice bath. To thisstirred mixture was added isoamyl nitrite (IAN, 1.5 eq., 215 μL, 1.6mmol) followed by trimethylsilyl azide (1.2 eq., 147 mg, 0.168 mL, 1.28mmol) dropwise. The resulting solution was stirred at room temperaturefor 45 minutes. The reaction mixture was concentrated under vacuum andthe crude product was resolubilised in EtOAc, washed with water, driedand evaporated to give 43 (177 mg, 1.06 mmol, 99%).

¹H (400 MHz, ACETONITRILE-d₃) δ 7.58-7.81 (m, J=8.78 Hz, 2H), 7.11-7.26(m, J=8.78 Hz, 2H); ¹³C NMR (101 MHz, ACETONITRILE-d₃) δ 144.9, 136.0,120.4, 113.6, 105.9, 83.4, 62.9; GC-ESI-MS: C₉H₅N₄ ⁺ [M+H]⁺, 169.0;found 169.0.

5-Azido-N-(4-(cyanoethynyl)phenyl)pentanamide (44)

44a: 5-Azidopentanoyl chloride

5-azidopentanoic acid (1 eq., 1.1 g, 6.99 mmol) was refluxed in SOCl₂(10 eq., 5.1 mL, 69.9 mmol) for 30 minutes. Excess of SOCl₂ was removedin vacuo and the obtained crude solid was used in the next step withoutpurification.

44: 5-Azido-N-(4-(cyanoethynyl)phenyl)pentanamide

7 (1 eq., 16.1 mg, 0.113 mmol) and TEA (1.5 eq., 24 μL, 0.17 mmol) weredissolved in DCM (3 mL), cooled to −78° C., and 44a (1.1 eq., 20.1 mg,0.125 mmol) was added to the reaction mixture that was then left to warmto room temperature while stirring for another 1 hour. The reaction masswas washed with 1M HCl (2×1 mL), water (2 mL), dried over Na₂SO₄, andevaporated to give crude product, which was purified by flashchromatography to give 44 (25.5 mg, 0.101 mmol, 89%) as a grey solid.

¹H NMR (400 MHz, METHANOL-d₄) δ 7.58-7.67 (m, J=8.70 Hz, 2H), 7.40-7.58(m, J=8.70 Hz, 2H), 3.23-3.28 (m, 2H), 2.34 (t, J=7.28 Hz, 2H),1.61-1.72 (m, 2H), 1.49-1.61 (m, 2H); ¹³C NMR (101 MHz, METHANOL-d₄) δ173.0, 144.0, 135.7, 134.3, 120.7, 113.0, 106.2, 84.5, 40.4, 34.4, 28.8,24.0; ESI-MS: C₁₄H₁₄N₅O⁺ [M+H]⁺, 268.1; found 268.1.

1-[4-(Cyanoethynyl)benzyl]-3,3,6,6-tetramethyl-4,5-didehydro-2,3,6,7-tetrahydrothiepiniumtriflate (45)

45c: 3-(p-Tolyl)prop-2-yn-1-ol

Synthesised using protocol A for Sonogashira coupling. Yellowish solid,yield: 88%.

¹H NMR (400 MHz, ACETONITRILE-d₃) δ 7.25-7.49 (m, J=8.03 Hz, 2H),7.04-7.25 (m, J=8.03 Hz, 2H), 4.34 (d, J=6.02 Hz, 2H), 3.31 (t, J=6.02Hz, 1H), 2.32 (s, 3H); ¹³C NMR (101 MHz, ACETONITRILE-d₃) δ 139.8,132.4, 130.3, 120.8, 88.8, 85.1, 51.2, 21.5; ESI-MS: C₁₀H₁₁O⁺ [M+H]⁺,146.1; found 146.0.

45b: 3-(p-Tolyl)propiolonitrile

The compound was obtained as the only product of the standard MnO₂oxidation protocol. Reaction time: 3 hours. White solid, yield: 67%.

¹H NMR (400 MHz, METHANOL-d₄) δ 7.37-7.59 (m, J=8.03 Hz, 2H), 7.02-7.31(m, J=8.03 Hz, 2H), 2.29 (s, 3H); ¹³C NMR (101 MHz, METHANOL-d₄) 143.2,133.3, 129.5, 114.0, 104.8, 83.2, 61.3, 20.4; ESI-MS: C₁₀H₈N⁻ [M+H]⁺,141.1; found 141.0.

45a: 3-(4-(Bromomethyl)phenyl)propiolonitrile

Degassed solution of 45b (1 eq., 68 mg, 0.482 mmol) in DCM (1 mL) wasMW-irradiated (100° C.) for 5 minutes. The reaction mixture wasevaporated, the crude was purified by preparative HPLC to give 45a (42.4mg, 0.193 mmol, 40%) as a yellowish solid.

¹H NMR (400 MHz, CHLOROFORM-d) δ 7.56-7.71 (m, J=8.28 Hz, 2H), 7.40-7.49(m, J=8.28 Hz, 2H), 4.48 (s, 2H); ¹³C NMR (101 MHz, CHLOROFORM-d) 141.8,133.9, 129.5, 117.5, 105.3, 82.3, 63.7, 31.8; GC-ESI-MS: C₁₀H₇BrN⁺[M+H]⁺, 219.0; found 219.0.

45:1-[4-(Cyanoethynyl)benzyl]-3,3,6,6-tetramethyl-4,5-didehydro-2,3,6,7-tetrahydrothiepiniumtriflate

To a degassed solution of 45a (1 eq., 43.7 mg, 0.199 mmol) and TMTH(1.29 eq., 43 mg, 0.255 mmol; synthesised following previously describedprocedure⁸³¹) in DCM (1.34 mL), a solution of LiOTf (11.6 eq., 360 mg,2.31 mmol) in distilled and degassed H₂O (0.668 mL) was added. Theobtained biphasic mixture was vigorously stirred for 5 days at 25° C.(degassing once per day). Two phases were separated, the organic one waswashed with DCM (5×2 mL). United organic fractions were evaporated andthe crude was purified by HPLC to give 45 (46.9 mg, 0.111 mmol, 56%) ascolourless oil (crystallizes slowly at 0° C. to yield a white solid).

¹H NMR (400 MHz, CHLOROFORM-d) δ 7.65-7.73 (m, J=8.03 Hz, 2H), 7.56-7.65(m, J=8.03 Hz, 2H), 5.07 (s, 2H), 4.12 (d, J=12.30 Hz, 2H), 3.72 (d,J=12.30 Hz, 2H), 1.36 (s, 6H), 1.30 (s, 6H); ¹³C NMR (101 MHz, DMSO-d₆)δ 135.1, 133.6, 131.9, 117.8, 106.4, 105.8, 83.3, 63.6, 60.1, 43.2,34.6, 26.4, 25.4; HR-ESI-MS: C₂₀H₂₂NS⁺ [M]⁺, 308.1; found 308.1.

Compounds 43-45 can be used for a click chemistry (such as reactionclick-azide) according to the invention.

Compound 45 can be used for strain promoted click according to theinvention.

1-({4-[1-{[2-({3-Carboxylato-4-[6-(dimethylamino)-3-(dimethyliminiumyl)-3H-xanthen-9-yl]phenyl}formamido)ethyl]sulfanyl}-2-cyanoeth-1-en-1-yl]phenyl}methyl)-3,3,6,6-tetramethyl-1-thiacyclohept-4-yn-1-iumtrifluoroacetate (46)

46a:2-(6-(Dimethylamino)-3-(dimethyliminio)-3H-xanthen-9-yl)-5-((2-mercaptoethyl)carbamoyl)benzoate

To a solution of TAMRA-5′-COOH (1 eq., 68.3 mg, 0.159 mmol) in DMF(0.228 mL), HATU (1 eq., 60.3 mg, 0.159 mmol), DIPEA (6 eq., 123 mg,0.157 mL, 0.952 mmol) and cystamine dichloride (5 eq., 178 mg, 0.793mmol) were subsequently added; the obtained solution mass was stirredovernight. A solution of DTT (5 eq., 122 mg, 0.118 mL, 0.793 mmol) inDCM (0.911 mL) was added to the reaction mass, the stirring continuedfor 2 hours. Solvents were evaporated; the obtained crude mass waspurified by HPLC to yield 46a (33.5 mg, 0.0555 mmol, 35%) as adark-violet solid.

¹H NMR (400 MHz, DMSO-d₆) δ 9.06 (t, J=5.4 Hz, 1H, 8.70 (d, J=1.8 Hz,1H), 8.30 (dd, J=1.8, 8.0 Hz, 1H), 7.59 (d, J=8.0 Hz, 1H), 7.08-7.02 (m,4H), 6.95 (s, 2H), 3.52-3.42 (m, 2H), 3.26 (s, 12H), 2.72 (dt, J=6.8,8.0 Hz, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 166.0, 164.7, 156.8, 156.6,135.9, 131.2, 130.6, 114.6, 96.3, 42.9, 40.5, 23.3; HR-ESI-MS:C₂₇H₂₇N₃O₄S: 489.1722; found 489.1723.

46:1-({4-[(1Z)-1-{[2-({3-Carboxylato-4-[6-(dimethylamino)-3-(dimethyliminiumyl)-3H-xanthen-9-yl]phenyl}formamido)ethyl]sulfanyl}-2-cyanoeth-1-en-1-yl]phenyl}methyl)-3,3,6,6-tetramethyl-1-thiacyclohept-4-yn-1-iumtrifluoroacetate (TAMRA-APN-TMTI)

A solution of 45 (1 eq., 6.74 mg, 0.016 mmol) in ACN (1 mL) was mixedwith a solution of 46a (1 eq., 9.64 mg, 0.016 mmol) in DMF (1 mL). DIPEA(5 eq., 132 μL, 0.08 mmol) was then added and obtained reaction mass wasinjected into HPLC after 5 minutes of reaction to yield 46 (11.9 mg,0.0149 mmol, 93%) as a dark-violet solid.

¹H NMR (400 MHz, DMSO-d₆) δ 8.98 (t, J=5.40 Hz, 1H), 8.30 (d, J=8.28 Hz,1H), 8.10-8.20 (m, 1H), 7.85 (s, 1H), 7.57-7.69 (m, 4H), 6.97-7.15 (m,5H), 6.07 (s, 1H), 4.85 (s, 2H), 2.81-2.90 (m, 4H), 3.28 (br. s., 16H),1.25 (s, 6H), 1.05 (s, 6H); ¹³C NMR (101 MHz, DMSO-d₆)—not informative(low resolved signals); HR-ESI-MS: C₄₇H₄₉N₄O₄S₂ ⁺, 797.31897; found797.32739.

1-({4-[2-Cyano-1-[(2-{4-[(E)-2-[4-(dimethylamino)phenyl]diazen-1-yl]benzenesulfonamido}ethyl)sulfanyl]eth-1-en-1-yl]phenyl}methyl)-3,3,6,6-tetramethyl-1-thiacyclohept-4-yn-1-ium(47, BHQ2-APN-TMTI)

47a:(E)-4-((4-(Dimethylamino)phenyl)diazenyl)-N-(2-mercaptoethyl)benzenesulfonamide

To a cooled to 0° C. solution of Dabsyl chloride (1 eq., 100 mg, 0.309mmol) in dry ACN (3 mL), TEA (7 eq., 218 mg, 0.3 mL, 2.16 mmol) andcystamine dihydrochloride (5 eq., 347 mg, 1.54 mmol) were subsequentlyadded. After 2 hours of stirring, DTT (6 eq., 285 mg, 0.275 mL, 1.85mmol) was added to the reaction mass. The obtained solution was stirredfor another 2 hours, evaporated and the obtained crude product waspurified by flash chromatography (cyclohexane-EtOAc) to yield 47a (105.9mg, 94%) as an orange solid.

47:1-({4-[(2-Cyano-1-[(2-{4-[(E)-2-[4-(dimethylamino)phenyl]diazen-1-yl]benzenesulfonamido}ethyl)sulfanyl]eth-1-en-1-yl]phenyl}methyl)-3,3,6,6-tetramethyl-1-thiacyclohept-4-yn-1-iumtrifluoroacetate

The same procedure as for the synthesis of the 46. Yield: 94%.

¹H NMR (400 MHz, DMSO-d₆) δ 8.03 (t, J=4.89 Hz, 1H), 7.91 (d, J=8.53 Hz,2H), 7.80-7.87 (m, J=9.04 Hz, 2H), 7.72-7.79 (m, J=8.53 Hz, 2H), 7.68(s, 4H), 6.87 (d, J=9.04 Hz, 2H), 6.08 (s, 1H), 4.86 (s, 2H), 3.92 (d,J=12.05 Hz, 2H), 3.84 (d, J=12.30 Hz, 2H), 3.10 (s, 6H), 2.71-2.87 (m,4H), 1.32 (s, 6H), 1.17 (s, 6H); ¹³C NMR (101 MHz, DMSO-d₆) δ 60.2,158.6, 158.3, 155.1, 153.7, 143.1, 140.3, 136.8, 131.9, 131.3, 129.5,128.2, 125.9, 122.8, 117.2, 112.1, 106.4, 99.4, 60.0, 43.3, 42.9, 34.5,26.4, 25.3; HR-ESI-MS: C₃₆H₄₂N₅O₂S₃ ⁺, 672.24951; found 672.25042.

Compounds 46-47 can be used for the preparation of compounds otherwisenot accessible (TMTI).

5-(3-{4-[1-(4-{[4-(2-Cyanoeth-1-yn-1-yl)phenyl]carbamoyl}butyl)-1H-1,2,3-triazol-4-yl]butanamido}propyl)-2-[(E)-2-[4-hydroxy-2-(2-{2-[2-(2-{5-[(4S)-2-oxo-hexahydro-1H-thieno[3,4-d]imidazolidin-4-yl]pentanamido}ethoxy)ethoxy]ethoxy}ethoxy)phenyl]diazen-1-yl]benzoic(48, APN-HAZA-biotin)

Compound 48 can be used for purification and/or immobilization accordingto the invention.

48a:2-(6-(Dimethylamino)-3-(dimethyliminio)-3H-xanthen-9-yl)-5-((2-mercaptoethyl)carbamoyl)benzoate

This compound was synthesised following the previously reportedprotocol.

48:5-(3-{4-[1-(4-{[4-(2-Cyanoeth-1-yn-1-yl)phenyl]carbamoyl}butyl)-1H-1,2,3-triazol-4-yl]butanamido}propyl)-2-[(E)-2-[4-hydroxy-2-(2-{2-[2-(2-{5-[(4S)-2-oxo-hexahydro-1H-thieno[3,4-d]imidazolidin-4-yl]pentanamido}ethoxy)ethoxy]ethoxy}ethoxy)phenyl]diazen-1-yl]benzoic(APN-HAZA-biotin)

To a solution of 48a (1 eq., 10 mg, 0.0123 mmol) and 77 (1 eq., 3.12 mg,0.0123 mmol) in DMSO (0.472 mL), solution of sodium ascorbate (10 eq.,24.4 mg, 0.123 mmol) and CuSO₄.5H₂O (5 eq., 15.4 mg, 0.0617 mmol) inwater was added. The obtained reaction mass was degassed and stirredovernight at 25° C. The reaction mass was directly purified by HPLC togive 48 (8.3 mg, 0.0078 mmol, 63%) as a yellow solid.

¹H NMR (400 MHz, METHANOL-d₄) δ 7.78 (br. s., 2H), 7.72 (d, J=8.28 Hz,1H), 7.67 (s, 1H), 7.58 (d, J=8.78 Hz, 2H), 7.50 (d, J=8.78 Hz, 2H),7.29 (d, J=8.28 Hz, 1H), 7.25 (d, J=8.53 Hz, 1H), 6.29 (d, J=7.78 Hz,1H), 3.70-3.81 (m, 8H), 3.62 (d, J=4.77 Hz, 2H), 3.58 (d, J=5.02 Hz,2H), 3.48-3.53 (m, 2H), 3.41-3.48 (m, 2H), 3.01-3.07 (m, 6H), 2.89-3.00(m, 10H), 2.78 (dd, J=4.89, 12.93 Hz, 1H), 2.52-2.67 (m, 8H), 2.33 (t,J=7.28 Hz, 2H), 2.09-2.19 (m, 2H), 1.81-1.91 (m, 4H), 1.65-1.78 (m, 2H),1.52-1.63 (m, 2H), 1.42-1.52 (m, 1H), 1.23-1.31 (m, 1H); HR-ESI-MS:C₅₆H₆₇N₁₁O₁₁S, 1077.47422; found 1077.45931.

2-((4-(cyanoethynyl)phenyl)amino)-N,N,N-trimethyl-2-oxoethan-1-aminiumtrifluoroacetate (49)

49a: 2-chloro-N,N,N-trimethyl-2-oxoethan-1-aminium

Synthesised as previously described by Vassel and Skelly(10.1002/0471264180.os035.09).

49:2-((4-(cyanoethynyl)phenyl)amino)-N,N,N-trimethyl-2-oxoethan-1-aminiumtrifluoroacetate

To a solution of 3-(4-aminophenyl)prop-2-ynenitrile (1 eq., 66.3 mg,0.466 mmol) and DIPEA (1.1 eq., 66.3 mg, 0.0848 mL, 0.513 mmol) in DMF(1 mL), a cooled to −20° C. solution of(2-chloro-2-oxoethyl)trimethylazanium chloride (1.1 eq., 88.3 mg, 0.513mmol) in DMF (1 mL) was added. The obtained reaction mass was stirred at25° C. for 10 hours, purified by RP-flash chromatography to give 49 as ayellowish solid (39 mg, 0.110 mmol, 24%).

¹H NMR (400 MHz, ACETONITRILE-d₃) δ 11.14 (br. s., 1H), 7.70-7.83 (m,J=8.78 Hz, 2H), 7.56-7.70 (m, J=8.78 Hz, 2H), 4.33 (s, 2H), 3.28 (s,9H); ¹³C NMR (101 MHz, ACETONITRILE-d₃) δ 163.5, 142.5, 135.8, 121.2,113.5, 106.5, 84.4, 66.3, 63.1, 55.2; ESI-MS: C₁₄H₁₆N₃O⁺ [M]⁺, 242.13;found 242.13.

3-(1-(1-(4-(cyanoethynyl)phenyl)-1H-1,2,3-triazol-4-yl)-2,5,8,11,14-pentaoxaheptadecan-17-amido)pentanedioicacid (50)

A solution of 43 (1 eq., 5.65 mg, 0.0336 mmol) in DMSO (0.0331 mL), asolution of di-acid-alkyne (1 eq., 14.6 mg, 0.0336 mmol) in water(0.0331 mL) was added. A solution of copper sulfate pentahydrate (0.1eq., 0.839 mg, 0.00336 mmol) in minimum amount of water was added to theobtained reaction mass followed by the addition of a solution of sodiumascorbate (0.5 eq., 3.33 mg, 0.0168 mmol) in minimum amount of water.The addition repeated after 30 minutes until complete disappearance ofthe strasting material (2 times overall). Excess of water was evaporatedto vacuo (no heating should be used, otherwise hydrolysis product startsto appear), the obtained crude mass was purified by HPLC after thefiltration of copper salts through a seringe filter to give 50 (11 mg,0.01828 mmol, 54%) as a white solid.

¹H NMR (400 MHz, ACETONITRILE-d₃) δ 8.41 (s, 1H), 7.95-8.02 (m, 2H),7.88-7.94 (m, 2H), 6.86 (d, J=7.78 Hz, 1H), 4.71 (s, 2H), 4.49 (td,J=6.71, 8.41 Hz, 1H), 3.68-3.76 (m, 3H), 3.49-3.68 (m, 21H), 2.53-2.64(m, 5H), 1.97 (td, J=2.42, 4.96 Hz, 15H); ¹³C NMR (101 MHz,ACETONITRILE-d₃) δ 188.7, 173.3, 161.6, 146.5, 135.1, 131.3, 130.8,122.5, 121.0, 117.9, 115.4, 64.2, 40.4, 39.5, 36.7, 30.6; ESI-MS:C₂₈H₃₄N₅O₁₀ ⁻ [M−H]⁻, 600.21; found 600.23.

N1,N5-bis(23-amino-3,6,9,12,15,18,21-heptaoxatricosyl)-3-(1-(1-(4-(cyanoethynyl)phenyl)-1H-1,2,3-triazol-4-yl)-2,5,8,11,14-pentaoxaheptadecan-17-amido)pentanediamide(51)

A solution of 43 (1 eq., 1.64 mg, 0.00973 mmol) and PEG-Alkyne (1 eq.,11 mg, 0.00973 mmol) in DMSO was added to a mixture of DMSO (0.00958 mL)and water (0.00958 mL). A solution of copper sulfate pentahydrate (0.1eq., 0.243 mg, 0.000973 mmol) in minimum amount of water was added tothe obtained reaction mass followed by the addition of a solution ofsodium ascorbate (0.5 eq., 0.964 mg, 0.00487 mmol) in minimum amount ofwater. The reaction mass was filtered and purified by HPLC to give 51 (5mg, 0.003839 mmol, 39%) as a colorless liquid.

ESI-HRMS: C₆₀H₁₀₃N₉O₂₂, 1301.72177; found 1301.72204.

N-(17-amino-3,6,9,12,15-pentaoxaheptadecyl)-1-(1-(4-(cyanoethynyl)phenyl)-1H-1,2,3-triazol-4-yl)-2,5,8,11,14-pentaoxaheptadecan-17-amide(52)

A solution of 3-(4-azidophenyl)prop-2-ynenitrile (1 eq., 14.9 mg, 0.0886mmol) in DMSO (0.0872 mL), a solution of PEG-alkyne (1 eq., 50.2 mg,0.0886 mmol) in water (0.0872 mL). To the obtained mixture a solution ofcopper sulfate pentahydrate (0.1 eq., 2.21 mg, 0.00886 mmol) in minimumamount of water was added followed by the addition of a solution ofsodium ascorbate (0.5 eq., 8.77 mg, 0.0443 mmol) in minimum amount ofwater. The addition of copper sulfate pentahydrate (0.1 eq., 2.21 mg,0.00886 mmol) and sodium ascorbate (0.5 eq., 8.77 mg, 0.0443 mmol) wererepeated after 30 minutes if starting azide was still present. Excess ofwater was evaporated to vacuo, the crude product was purified by HPLC togive 52 (55 mg, 0.07485 mmol, 84%) as a colorless oil.

¹H NMR (400 MHz, ACETONITRILE-d₃) δ 8.42 (s, 1H), 7.95-8.02 (m, J=9.03Hz, 2H), 7.86-7.95 (m, J=8.78 Hz, 2H), 7.30 (br. s., 2H), 7.19 (br. s.,1H), 4.71 (s, 2H), 3.73-3.79 (m, 2H), 3.65-3.73 (m, 7H), 3.54-3.65 (m,29H), 3.51 (t, J=5.40 Hz, 2H), 3.34 (q, J=5.35 Hz, 2H), 3.13 (d, J=4.52Hz, 2H), 2.37-2.46 (m, 2H), 1.92-2.01 (m, 5H); ¹³C NMR (126 MHz,CHLOROFORM-d) δ 177.5, 151.3, 144.6, 140.7, 140.7, 140.6, 127.2, 126.0,125.9, 125.8, 122.4, 110.5, 87.2, 75.4, 75.3, 75.3, 75.2, 75.2, 75.1,75.1, 75.1, 75.0, 74.9, 74.8, 74.8, 72.2, 71.9, 68.9, 68.5, 45.0, 44.2,41.6; ESI-MS: C₃₅H₅₅N₆O₁₁ ⁺ [M+H]⁺, 735.39; found 735.20.

1-(4-(cyanoethynyl)phenyl)-3-(3-(dimethylamino)propyl)urea (53)

To a solution of triphosgene (1 eq., 56.3 mg, 31.6 μL, 0.19 mmol) in THF(0.404 μL) was added a solution of 3-(4-aminophenyl)prop-2-ynenitrile (3eq., 80.9 mg, 0.569 mmol) in THF (0.404 μL). Then triethylamine (6 eq.,115 mg, 158 μL, 1.14 mmol). The mixture was stirred for 5 min and then3-dimethylaminopropylamine (3 eq., 58.1 mg, 71.8 μL, 0.569 mmol) andtriethylamine (2 eq., 38.4 mg, 52.7 μL, 0.379 mmol) was added in THF(0.404 μL). The reaction mixture was stirred for 10 minutes and thenconcentrated. The obtained residue was purified by HPLC to give 53 (47mg, 0.1764 mmol, 93%) as a white solid.

¹H NMR (400 MHz, METHANOL-d₄) δ 7.45-7.50 (m, 2H), 7.41-7.45 (m, 2H),3.23-3.25 (m, 1H), 3.04-3.11 (m, 2H), 2.80 (s, 6H), 1.77-1.91 (m, 2H);¹³C NMR (101 MHz, METHANOL-d₄) δ 156.5, 143.7, 134.4, 118.0, 109.3,105.0, 83.7, 61.0, 55.2, 42.1, 35.9, 25.3; ESI-MS: C₁₅H₂₀N₄O⁺ [M+H]⁺,271.16; found 271.15.

3-((3-(3-(4-(cyanoethynyl)phenyl)ureido)propyl)dimethylammonio)propane-1-sulfonate(54)

53 (1 eq., 27 mg, 0.0702 mmol) and 1,3-propanesultone (1.1 eq., 9.44 mg,0.00678 mL, 0.0773 mmol) were dissolved in IPA (0.5 mL) and refluxed for3 h. The reaction mixture was cooled to room temperature and theprecipitate was filtered and washed with cold distilled water to removeany unreacted propanesultone to give 54 (27 mg, 0.0688 mmol, 98%) as awhite solid.

¹H NMR (400 MHz, DEUTERIUM OXIDE) δ 7.45-7.67 (m, 2H), 7.33 (br. s.,2H), 3.61 (br. s., 4H), 3.53 (br. s., 1H), 3.39 (br. s., 2H), 3.31 (br.s., 2H), 3.25 (br. s., 2H), 3.03 (br. s., 7H), 2.89 (br. s., 7H), 2.13(br. s., 1H), 1.89 (br. s., 7H), 1.08 (br. s., 2H); ESI-HRMS:C₁₈H₂₄N₄O₄S, 392.15183; found 392.15254. Compounds 49-54 can be used fora conjugation method according to the invention, for instance forchanging ADME parameters (solubilizing agents).

3-(4-(4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)phenyl)propiolonitrile(55)

3-(4-azidophenyl)prop-2-ynenitrile (1 eq., 300 mg, 1.78 mmol),2-propyn-1-ol (2 eq., 200 mg, 0.211 mL, 3.57 mmol) were solubilized inTHF (9 mL). To this mixture was added a solution of copper sulfatepentahydrate (10%, 44.5 mg, 0.178 mmol) in 1.5 mL of water followed bythe solution of sodium ascorbate (0.5 eq., 176 mg, 0.892 mmol) in 1.5 mLof water. The resulting solution was stirred for 2 h and thenconcentrated on rotary evaporator. The residue was extracted with DCM.The organic layer was washed with NH₄Cl (sat.) and water, dried overMgSO₄ and then evaporated. The residue was resolubilised in DCM and theproduct was filtered to give 55 (23.28 mg, 0.08108 mmol, 92%) as aslightly yellowish solid.

1H NMR (400 MHz, MeOD) δ 8.58 (s, 1H), 8.04 (d, J=7.4 Hz, 2H), 7.93 (d,J=7.4 Hz, 2H), 4.77 (s, 2H); ESI-MS: C₁₂H₉N₄O⁺ [M+H]⁺, 225.08; found225.05.

3-(4-(4-(bromomethyl)-1H-1,2,3-triazol-1-yl)phenyl)propiolonitrile (56)

55 (1 eq., 19.8 mg, 0.0881 mmol) was dissolved in THF (1 mL) undernitrogen at room temperature and3-{4-[4-(bromomethyl)-1H-1,2,3-triazol-1-yl]phenyl}prop-2-ynenitrile(23.3 mg, 0.0811 mmol, 92%) was added. The reaction mixture was stirredat room temperature for 15 hours. Solvents were evaporated, the crudeproduct was purified by HPLC to give 56 (23.3 mg, 0.0811 mmol, 92%) as awhite solid.

¹H NMR (400 MHz, ACETONITRILE-d₃) δ 8.44 (s, 1H), 7.78-8.03 (m, 5H),4.74 (s, 2H); ¹³C NMR (101 MHz, CHLOROFORM-d) δ 150.9, 144.3, 140.6,140.5, 126.0, 125.9, 110.4, 87.1, 68.5, 27.0; ESI-MS: C₁₂H₈BrN₄ ⁺[M+H]⁺, 286.99; found 287.08.

Compound 56 can be used for a bioconjugation method according to theinvention.

1-({1-[4-(cyanoethynyl)phenyl]-1H-1,2,3-triazol-4-yl}methyl)-3,3,6,6-tetramethyl-4,5-didehydro-2,3,6,7-tetrahydrothiepiniumtrifluoroacetate (57)

To a degassed solution of3-{4-[4-(bromomethyl)-1H-1,2,3-triazol-1-yl]phenyl}prop-2-ynenitrile (1eq., 19.6 mg, 0.0684 mmol) and TMTH (1.89 eq., 21.7 mg, 0.129 mmol) inDCM (0.982 mL), a solution of lithium triflate (10 eq., 106 mg, 0.684mmol) in water (0.982 mL) was added. The obtained biphasic mixture wasvigorously mixed for 2 days at 25° C. Phases were separated, organicphase was washed with DCM (5×2 mL).

United organic fractions were evaporated and the crude was purified byHPLC to give 57 (23.7 mg, 0.0451, 66%) as a white solid.

ESI-HRMS: C₂₂H₂₃N₄S, 375.16434; found 375.16497.

Compound 57 can be used for click-chemistry (strain promoted click)according to the invention.

4-(((1-(4-(cyanoethynyl)phenyl)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl)-2-(6-(dimethylamino)-3-(dimethyliminio)-3H-xanthen-9-yl)benzoate(58)

2-[6-(dimethylamino)-3-(dimethyliminiumyl)-3H-xanthen-9-yl]-4-[(prop-2-yn-1-yl)carbamoyl]benzoate(1 eq., 52.8 mg, 0.113 mmol) and 3-(4-azidophenyl)prop-2-ynenitrile (1eq., 19 mg, 0.113 mmol) were solubilized in THF (1 mL). H₂O (1 mL) wasadded to the obtained reaction mixture followed by the addition ofsolutions of Copper Sulphate pentahydrate (10%, 2.82 mg, 0.0113 mmol)and sodium ascorbate (50%, 11.2 mg, 0.0565 mmol) in minimum amount ofwater (separately). The obtained reaction mixture was stirred foranother 30 minutes, evaporated and purified by HPLC to give 58 (66.8 mg,0.105 mmol, 93%) as a dark-violet solid.

¹H NMR (400 MHz, METHANOL-d₄) δ 8.51 (s, 1H), 8.33 (d, J=8.28 Hz, 1H),8.16 (dd, J=1.51, 8.28 Hz, 1H), 7.86-8.02 (m, 2H), 7.64-7.86 (m, 3H),7.02-7.12 (m, 2H), 6.84-7.02 (m, 4H), 4.58-4.71 (m, 2H); ESI-HRMS:C₃₇H₂₉N₇O₄, 635.22811; found 635.22861.

3-(9-(diethylamino)-5-oxo-5H-benzo[a]phenoxazin-2-yl)propiolonitrile(59)

59b: 8-(diethylamino)-3-hydroxy-12H-10-oxa-5-azatetraphen-12-one (1 eq.,65 mg, 0.194 mmol) was dissolved in dry DCM (2 mL) and cooled to 5° C.Then TEA (1.2 eq., 23.6 mg, 0.0324 mL, 0.233 mmol) was added followed bythe addition of TfCl (1.2 eq., 39.3 mg, 0.0249 mL, 0.233 mmol). Theaddition of Tf2O was repeated untile complete disappearance of thestarting material. The solvent was evaporated and the residue wastreated with water. The precipitate was filtered off and washed withwater and heptane to give the desired product (76 mg, 0.163 mmol, 84%)as a dark-violet solid.

ESI-HRMS: C₂₁H₁₇F₃N₂O₅S, 466.08103; found 466.08221.

59a: Under an inert atmosphere, DIPEA (2 eq., 16.1 mg, 0.0206 mL, 0.124mmol) and Propargylic alcohol (1.5 eq., 5.23 mg, 0.00551 mL, 0.0933mmol) were added to a solution of 59b (1 eq., 29 mg, 0.0622 mmol),PdCl₂(PPh₃)₂ (5%, 2.18 mg, 0.00311 mmol) and CuI (10%, 1.18 mg, 0.00622mmol) in DMF (1 mL). After stirring for 2 hours at 90° C., the solventwas removed under reduced pressure. The crude product was purified byflash chromatography (DCM-MeOH from 100-0 to 80-20).

ESI-HRMS: C₂₃H₂₀N₂O₃, 372.14739; found 372.14735.

59: To the solution of 59a (1 eq., 10 mg, 0.0269 mmol) in THF (0.121 mL)was added MgSO₄ (15 eq., 48.5 mg, 0.403 mmol), NH₃ (4 eq., 2 M, 0.0537mL, 0.107 mmol), and MnO₂ (15 eq., 35 mg, 0.403 mmol). The reactionmixture was stirred at r.t. for 15 mins and followed by HPLC. Aftercompletion the mixture was filtered through Celite and washed thoroughlywith THF. Evaporation of the filtrate gave crude 59 (9.47 mg, 0.0258mmol, 96%) which was purified by HPLC.

¹H NMR (400 MHz, CHLOROFORM-d) 68.91 (s, 1H), 8.26 (d, J=8.03 Hz, 1H),7.79 (dd, J=1.51, 8.03 Hz, 1H), 7.62 (d, J=9.29 Hz, 1H), 6.74 (dd,J=2.63, 9.16 Hz, 1H), 6.51 (d, J=2.51 Hz, 1H), 6.35 (s, 1H), 5.31 (s,2H), 3.49 (q, J=7.11 Hz, 4H), 1.09-1.36 (m, 6H); ESI-HRMS: C₂₃H₁₇N₃O₂,367.13208; found 367.13145.

(E)-3-(4-((4-(dimethylamino)phenyl)diazenyl)phenyl)propiolonitrile (60)

7 (1 eq., 167 mg, 1.17 mmol) was dissolved in acetonitrile (2.58 mL). Tothis stirred mixture was added Isoamyl nitrite (1.5 eq., 206 mg, 0.237mL, 1.76 mmol), stirring continued for another 2 minutes, thendimethylaniline (1.1 eq., 156 mg, 0.165 mL, 1.29 mmol) was added. Theresulting reaction mixture was stirred overnight (turned red),evaporated and purified by flash chromatography (DCM, first peak) togive 60 (120 mg, 0.437 mmol, 37%) as red solid.

¹H NMR (400 MHz, CHLOROFORM-d) δ 7.91 (d, J=9.03 Hz, 2H), 7.82-7.88 (m,J=8.53 Hz, 2H), 7.62-7.77 (m, J=8.53 Hz, 2H), 6.77 (d, J=9.03 Hz, 2H),3.08-3.18 (m, 6H); ¹³C NMR (101 MHz, CHLOROFORM-d) δ 154.7, 153.2,143.7, 134.4, 125.8, 122.5, 117.3, 111.6, 105.6, 83.3, 64.2, 40.3;ESI-HRMS: C₁₇H₁₄N₄, 274.12185; found 274.12247.

Compounds 58-60 can be used for a detection method according to theinvention.

tert-butyl((1-(4-(cyanoethynyl)phenyl)-1H-1,2,3-triazol-4-yl)methyl)carbamate(61)

To a solution of 43 (1 eq., 51.5 mg, 0.306 mmol) and boc-propargylamine(1 eq., 47.5 mg, 0.306 mmol) in THF (2 mL) were added H₂O (1 mL) andsolution of CuSO₄ (10%, 4.89 mg, 0.0306 mmol) and sodium ascorbate (50%,30.3 mg, 0.153 mmol) in water (50 uL each). Stirring continued for 10minutes, one more portion of CuSO₄ (10%, 4.89 mg, 0.0306 mmol) andsodium ascorbate (50%, 30.3 mg, 0.153 mmol) was added. After another 15minutes of stirring, Et₂O (15 mL) and NH₄Cl (sat, 10 mL) were added.Organic phase was washed two more times with NH₄Cl (sat, 10 mL), driedover MgSO₄ and evaporated to give 61 (98 mg, 0.303 mmol, 99%) as ayellow solid. Used without further purification.

¹H NMR (400 MHz, CHLOROFORM-d) δ 7.97 (s, 1H), 7.74-7.81 (m, J=8.78 Hz,2H), 7.67-7.74 (m, J=8.78 Hz, 2H), 4.41 (d, J=6.02 Hz, 2H), 1.32-1.41(m, 9H); ¹³C NMR (101 MHz, CHLOROFORM-d) δ 139.0, 135.1, 120.4, 117.8,107.2, 105.1, 81.3, 64.5, 28.4; ESI-MS: C₁₇H₁₈N₅O₂ ⁺ [M+H]⁺, 323.14;found 323.13.

(1-(4-(cyanoethynyl)phenyl)-1H-1,2,3-triazol-4-yl)methanaminiumtrifluoroacetate (62)

To a solution of 61 (1 eq., 21.5 mg, 0.0665 mmol) in DCM (1 mL) wasadded TFA (20 eq., 151 mg, 0.0988 mL, 1.33 mmol). The obtained reactionmixture was left overnight at room temperature (or 2 hours at 37° C.) tothe targeted product (22.4 mg, 0.0665 mmol, 100%) after the evaporationof all volatile compounds.

¹H NMR (400 MHz, METHANOL-d₄) 68.74 (s, 1H), 7.99-8.15 (m, 2H),7.85-7.97 (m, 2H), 4.25-4.45 (m, 2H); ¹³C NMR (101 MHz, METHANOL-d₄) δ141.2, 138.9, 135.2, 122.3, 120.3, 117.7, 104.4, 81.1, 62.9, 34.0;ESI-MS: C₁₂H₁₀N₅ ⁺ [M]⁺, 227.09; found 227.10.

N-((1-(4-(cyanoethynyl)phenyl)-1H-1,2,3-triazol-4-yl)methyl)-4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzamide(63)

A solution of 4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzoic acid (1eq., 57.8 mg, 0.251 mmol), HATU (1 eq., 95.5 mg, 0.251 mmol), and DIPEA(3 eq., 97.4 mg, 0.125 mL, 0.753 mmol) in DMF (2 mL) was added onto 62(1 eq., 84.7 mg, 0.251 mmol). The obtained reaction mass was stirred for10 minutes and purified by HPLC to give the targeted compound (76.5 mg,0.176 mmol, 70%) as a white solid.

ESI-HRMS: C₂₁H₁₂F₃N₇O, 435.10554; found 435.10512.

Compound 63 can be used for a labeling method according to theinvention, such as for photolabeling of proteins.

tert-Butyl(2-((2-(4-(cyanoethynyl)benzamido)ethyl)disulfanyl)ethyl)carbamate(64)

To a solution of 23 (1 eq., 36 mg, 0.143 mmol) in acn (1 mL) was added asolution of tert-butyl N-{2-[(2-aminoethyl)disulfanyl]ethyl}carbamate (1eq., 36 mg, 0.143 mmol) and DIPEA (2.12 eq., 39.1 mg, 0.05 mL, 0.303mmol) in ACN (1 mL). The obtained reaction mixture was left for 10minutes, then solvents were evaporated and the crude product waspurified by flash chromatography to give the targeted product (29.3 mg,0.0723 mmol, 51%) as a yellowish solid.

¹H NMR (400 MHz, METHANOL-d₄) δ 7.74-7.84 (m, J=8.53 Hz, 2H), 7.58-7.74(m, J=8.53 Hz, 2H), 3.60 (t, J=6.78 Hz, 2H), 3.23-3.35 (m, 2H), 2.85 (t,J=6.78 Hz, 2H), 2.71 (t, J=6.90 Hz, 2H), 1.32 (s, 9H); ¹³C NMR (101 MHz,METHANOL-d₄) δ 167.3, 157.0, 137.3, 133.5, 127.5, 120.1, 104.4, 81.5,78.8, 63.1, 39.3, 39.1, 37.8, 27.4, 26.6; ESI-MS: C₁₉H₂₄N₃O₃S₂ ⁺ [M+H]⁺,406.12; found 406.10.

2-((2-(4-(cyanoethynyl)benzamido)ethyl)disulfanyl)ethan-1-aminiumtrifluoroacetate (65)

To a solution of 64 (1 eq., 29.3 mg, 0.0723 mmol) in ACN-DCM mixture (1mL of each solvent) was added TFA (10 eq., 82.4 mg, 0.0537 mL, 0.723mmol). The obtained reaction mixture was stirred for 24 hours, andevaporated to give the targeted compound 65 (30 mg, 0.0715 mmol, 99%) asa colorless liquid.

ESI-HRMS: C₁₄H₁₆N₃OS₂ ⁺, 306.07293; found 306.07312.

2-((2-(4-(cyanoethynyl)benzamido)ethyl)disulfanyl)ethan-1-aminiumtrifluoroacetate (66)

62 (0.567 eq., 10.1 mg, 0.0299 mmol) solution in MeOH (0.0881 mL) wasslowly added to a solution of2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]aceticacid (1 eq., 21.4 mg, 0.0528 mmol) in water (0.661 mL). The mixture wascooled by ice and pH was adjusted to 5 using DIPEA (25.2 eq., 172 mg,0.22 mL, 1.33 mmol). An aqueous solution of EDC (0.65 eq., 6.59 mg,0.0344 mmol) was added dropwise and stirred for 20 min with ice cooling.pH was raised to 8 using DIPEA and reacted for 30 min at roomtemperature. The end point of the reaction was monitored using HPLC.

ESI-HRMS: C₂₈H₃₅N₉P₇ ⁺, 609.26594; found 609.26417.

Compound 66 can be used as a chelating agent.

5-((4-((4-(2-Cyano-1-((2-(4-((4-((E)-(2,5-dimethoxy-4-((E)-(4-nitrophenyl)diazenyl)phenyl)diazenylphenyl)-(methyl)amino)butanamido)ethyl)thio)vinyl)phenyl)amino)-4-oxobutyl)carbamoyl)-2-(6-(dimethylamino)-3-(dimethyliminio)-3H-xanthen-9-yl)benzoate(A)

4-((4-((E)-(2,5-Dimethoxy-4-((E)-(4-nitrophenyl)diazenyl)phenyl)diazenyl)phenyl)(methyl)amino)-butanoicacid (BHQ-2)

Fast Black K hemi (zinc chloride) salt (practical grade, ≈30% dyecontent) (7.76 g) was suspended in cold water (150.0 mL, 0° C.) andstirred for 20 minutes. The suspension was filtered, and the redsolution was added dropwise to a cold (0° C.) mixture of4-(methyl(phenyl)amino)butanoic acid (1.33 g, 6.88 mmol), concentratedhydrochloric acid (3.1 mL) and sodium acetate (3.6 g, 43.90 mmol) inwater-acetone mixture (1:1) (150.0 mL). The reaction mixture was stirredat 10° C. for 15 minutes and at room temperature for 2 hours. Then thereaction crude was extracted with ethyl acetate (3×150 mL) and thecombined organic layers were dried over Na₂SO₄. The crude product waspurified by column chromatography on silica gel (100% EtOAc, then 100%DCM to DCM/MeOH (95:5)). BHQ-2 (1.36 g, 39%) was obtained as a darkviolet solid.

1H NMR (400 MHz, METHANOL-d₄) δ 8.31 (d, J=9.0 Hz, 2H), 8.00 (d, J=9.0Hz, 2H), 7.86 (d, J=9.0 Hz, 2H), 7.45 (s, 1H), 7.40 (s, 1H), 6.77 (d,J=9.0 Hz, 2H), 4.05 (s, 3H), 4.00 (s, 3H), 3.5 (t, J=7.1 Hz, 2H), 2.36(t, J=7.1 Hz, 2H), 1.98-1.90 (m, 2H); 13C NMR (101 MHz, METHANOL-d₄) δ176.2, 157.1, 154.3, 153.0, 151.4, 149.0, 147.4, 145.0, 142.6, 126.9,125.3, 124.2, 112.1, 101.7, 100.7, 57.2, 52.3, 39.0, 31.6, 22.9.

65b:4-((4-((E)-(2,5-Dimethoxy-4-((E)-(4-nitrophenyl)diazenyl)phenyl)diazenyl)phenyl)(methyl)amino)-N-(2-mercaptoethyl)butanamide((BHQ-2)-SH)

BHQ-2 (1 eq., 92.2 mg, 0.182 mmol) was dissolved in a mixture of DMF (5mL) and DCM (10 mL). TEA (6 eq., 152 μL, 1.09 mmol) and cystaminedichloride (5 eq., 204 mg, 0.91 mmol) were added. The mixture was cooledto 0° C. and HBTU (1 eq., 69 mg, 0.182 mmol) was added. The solution wasallowed to reach room temperature and stirred for 15 hours. When totalconversion was reached, DTT (6 eq., 168 mg, 0.162 mL, 1.09 mmol) wasadded. After the resulting mixture has been stirred for 10 minutes atroom temperature, the crude was diluted with saturated NaHCO₃ solution(75 mL) and extracted with EtOAc (2×50 mL). The organic layers werecombined, washed with water (50 mL), brine (50 mL) and dried overNa₂SO₄. The crude product was purified by column chromatography onsilica gel (DCM/MeOH from 100:0 to 95:5) to yield (BHQ-2)-SH (60.7 mg,0.107 mmol, 59%) as a dark violet solid.

¹H NMR (400 MHz, CHLOROFORM-d) δ 8.33 (d, J=9.0 Hz, 2H), 8.0 (d, J=9.1Hz, 2H), 7.9 (d, J=9.1 Hz, 2H), 7.42 (s, 1H), 7.42 (s, 1H), 6.75 (d,J=9.00 Hz, 2H), 5.90 (t, J=5.6 Hz, 1H), 4.06 (s, 3H), 4.01 (s, 3H), 3.49(t, J=7.4 Hz, 2H), 3.41 (dt, J=6.2, 6.4 Hz, 2H), 2.64 (td, J=6.4, 8.47Hz, 2H), 2.24 (t, J=7.4 Hz, 2H), 2.01-1.94 (m, 2H); 13C NMR (101 MHz,CHLOROFORM-d) δ 172.2, 156.6, 153.8, 152.4, 151.1, 148.5, 147.0, 144.7,142.3, 126.4, 124.9, 123.7, 111.6, 101.2, 100.3, 57.0, 56.9, 51.8, 42.5,38.7, 33.4, 24.9, 23.0; HR-ESI-MS: C₂₇H₃₁N₇O₅S, 565.2107; found565.2105.

67:5-((4-((4-(Cyanoethynyl)phenyl)amino)-4-oxobutyl)carbamoyl)-2-(6-(dimethylamino)-3-(dimethyliminio)-3H-xanthen-9-yl)benzoate

To a cooled to 0° C. degassed solution of 62a (1 eq., 17.3 mg, 0.0507mmol) and TAMRA-5′-COOH (1 eq., 21.8 mg, 0.0507 mmol) in DMF (1.4 mL),HBTU (1 eq., 19.2 mg) was added at 0° C. Obtained reaction mass wasstirred for 5 minutes and TEA was added. The reaction mass was stirredfor 1 hour at 25° C., evaporated and purified by HPLC to yield 65a (22mg, 68%) as a dark-violet solid.

¹H NMR (400 MHz, METHANOL-d₄) δ 8.8 (br. s, 1H), 8.7 (s, 1H), 8.08-8.16(d, J=8.2 Hz, 1H), 7.60-7.70 (d, J=8.9 Hz, 2H), 7.49-7.58 (d, J=8.9 Hz,2H), 7.32-7.39 (d, J=8.2, 1H), δ 7.01 (s, 4H), 6.93 (s, 2H), 3.48-3.58(m, 2H), 3.26 (s, 12H), 2.44-2.54 (t, J=7.17 Hz, 2H), 1.98-2.12 (m, 2H);¹³C NMR (101 MHz, METHANOL-d₄)—not informative; HR-ESI-MS: 639.24817;found 639.24310.

A:5-((4-((4-(2-Cyano-1-((2-(4-((4-((E)-(2,5-dimethoxy-4-((E)-(4-nitrophenyl)diazenyl)phenyl)diazenylphenyl)-(methyl)amino)butanamido)ethyl)thio)vinyl)phenyl)amino)-4-oxobutypcarbamoyl)-2-(6-(dimethylamino)-3-(dimethyliminio)-3H-xanthen-9-yl)benzoate

To a degassed solution of BHQ-SH (1.13 eq., 2 mg, 0.00354 mmol) in DCM(0.5 mL), a degassed solution of 67 (1 eq., 2 mg, 0.00313 mmol) inmethanol (0.5 mL) was added. TEA (4.6 eq., 2 μL, 0.0144 mmol) was addedand the obtained reaction mass was left overnight at 25° C. Solventswere evaporated; the crude product was solubilised in DMSO (0.5 mL) andpurified by HPLC to give BHQ-APN-TAMRA (A, 2.7 mg, 0.00225 mmol, 72%) asdark-violet solid.

¹H NMR (400 MHz, METHANOL-d₄) δ 8.63 (d, J=2.0 Hz, 1H), 8.34 (d, J=8.8Hz, 2H), 8.22 (dd, J=7.8, 2.0 Hz, 1H), 8.00 (d, J=8.8 Hz, 2H), 7.75 (d,J=9.0 Hz, 2H), 7.69 (d, J=8.5 Hz, 2H), 7.53 (d, J=8.0 Hz, 1H), 7.39 (d,J=8.5 Hz, 2H), 7.34 (d, J=9.0 Hz, 2H), 6.75-6.86 (m, 8H), 5.48 (s, 1H),4.02 (s, 3H), 3.92 (s, 3H), 3.58-3.63 (m, 2H), 3.45-3.52 (m, 2H), 3.21(s, 12H), 3.16 (t, J=6.9 Hz, 2H), 3.08 (s, 3H), 2.71 (t, J=6.5 Hz, 2H),2.55 (t, J=6.2 Hz, 2H), 2.22 (t, J=6.9 Hz, 2H), 2.08-2.16 (m, 2H),1.88-1.96 (m, 2H), 1.61 (br.s, 1H); ¹³C NMR (101 MHz, METHANOL-d₄)—notinformative; HR-ESI-MS: C₆₅H₆₅N₁₂O₁₀S⁺ [M+H]⁺, 1205.46618; found1205.46748.

5-((3-(3-((2-(4-((4-((E)-(2,5-Dimethoxy-4-((E)-(4-nitrophenyl)diazenyl)phenyl)diazenyl)phenyl)(methyl)amino)butanamido)ethyl)thio)-2,5-dioxopyrrolidin-1-yl)propyl)carbamoyl)-2-(6-(di-methylamino)-3-(dimethyliminio)-3H-xanthen-9-yl)benzoate(B)

B-4: 3a,4,7,7a-Tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione

This compound was synthesised according to the previously describedprocedure.⁸⁴⁵

B-3:tert-Butyl(3-((3aR,7aS)-1,3-dioxo-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindol-2(3H)-yl)propyl)carbamate

To the solution of B-4 (1 eq., 1.76 g, 10.7 mmol) and tert-butylN-(3-bromopropyl)carbamate (2 eq., 5.07 g, 21.3 mmol) in DMF (20 mL),K₂CO₃ (1.2 eq., 1.77 g, 12.8 mmol) was added. The obtained reaction masswas heated at 50° C. for 18 hours. The solution was left to cool down; asolid residue was filtered and washed with DMF. United organic fractionswere evaporated, hexane (50 mL) was added to the obtained slurry mass.Obtained suspensions were stirred for another hour, filtered and washedwith hexane to give B-3 (3.36 g, 10.4 mmol, 98%) as white solid.

¹H NMR (400 MHz, CHLOROFORM-d) δ 6.49 (s, 2H), 5.23 (s, 2H), 3.52 (t,J=6.5 Hz, 2H), 2.96-3.09 (m, 2H), 2.82 (s, 2H), 1.66-1.75 (m, 2H), 1.41(s, 9H); ¹³C NMR (101 MHz, CHLOROFORM-d) δ 176.5, 155.9, 136.5, 81.0,79.3, 47.5, 37.1, 36.0, 28.4, 27.8.

B-2: 1-(3-Aminopropyl)-1H-pyrrole-2,5-dione (TFA salt)

A solution of 66c (1 eq., 243 mg, 0.754 mmol) in toluene (25 mL) wasrefluxed for 3 hours. Toluene was evaporated; the obtained white crudeproduct was resolubilised in DCM (5 mL), TFA (0.5 mL) was added.Stirring was continued for 2 hours until complete disappearance of astarting material (controlled by TLC). Solvent were evaporated after thereaction was quenched by methanol (3 mL). Obtained1-(3-aminopropyl)-1H-pyrrole-2,5-dione (B-2, TFA salt, 190 mg, 94%) wasused without further purification.

¹H NMR (400 MHz, METHANOL-d₄) δ 6.76 (s, 2H), 3.52 (t, J=6.7 Hz, 2H),2.80-2.88 (m, 2H), 1.76-1.88 (m, 2H); ¹³C NMR (101 MHz, METHANOL-d₄) δ170.6, 135.6, 38.5, 35.4, 28.0.

B-1:2-(6-(Dimethylamino)-3-(dimethyliminio)-3H-xanthen-9-yl)-5-((3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propyl)carbamoyl)benzoate(TAMRA-maleimide)

To a solution of TAMRA-5′-COOH (1 eq., 71.5 mg, 0.166 mmol) in DMF (3.21mL), TEA (2.5 eq., 57.7 μL, 0.415 mmol) and HATU (1.12 eq., 70.7 mg,0.186 mmol) here added. Obtained reaction mass was stirred for another 5minutes and B-2 (1 eq., 71.5 mg, 0.166 mmol) was added. Stirringcontinued for 25 minutes and the reaction mass was evaporated underreduced pressure to the volume of about 1 mL, and the reaction mass waspurified by preparative HPLC to give TAMRA-Maleimide (B-1, 34.8 mg,0.0615 mmol, 37%) as a pink solid.

¹H NMR (400 MHz, METHANOL-d₄) δ 8.66 (d, J=1.8 Hz, 1H), 8.14 (dd, J=1.8,8.0 Hz, 2H), 7.41 (d, J=8.0 Hz, 2H), 7.02 (d, J=9.5 Hz, 1H), 6.92 (dd,J=9.5, 2.2 Hz, 2H), 6.81 (d, J=2.2 Hz, 2H), 6.72 (s, 2H), 3.52 (t, J=6.8Hz, 2H), 3.34 (t, J=7.0 Hz, 2H), 3.17 (s, 12H), 1.80-1.90 (m, 2H); ¹³CNMR (101 MHz, METHANOL-d₄) δ 172.5, 168.2, 167.4, 160.6, 159.0, 158.9,138.1, 137.7, 137.6, 135.5, 132.9, 132.3, 132.0, 131.4, 115.6, 114.8,97.5, 82.4, 41.0, 38.6, 36.4, 29.3. HR-ESI-MS: C₃₂H₃₀N₄O₆, 566.21653;found 566.21654.

B:5-((3-(3-((2-(4-((4-((E)-(2,5-Dimethoxy-4-((E)-(4-nitro-phenyl)diazenyl)phenyl)diazenyl)phenyl)(methyl)amino)butanamido)ethyl)thio)-2,5-dioxopyrrolidin-1-yl)propyl)carbamoyl)-2-(6-(dimethylamino)-3-(dimethyliminio)-3H-xanthen-9-yl)benzoate

To a degassed solution of BHQ-SH (1.15 eq., 4.6 mg, 0.00812 mmol) in DCM(0.5 mL), a degassed solution of TAMRA-Maleimide (B-1) (1 eq., 4 mg,0.00313 mmol) in methanol (0.5 mL) was added. TEA (5 eq., 5 μL, 0.0353mmol) was added and the obtained reaction mass was left overnight at 25°C. Solvents were evaporated; the crude product was solubilized in DMSO(0.5 mL) and purified by HPLC to give B (7 mg, 0.00621 mmol, 88%) asdark-violet solid.

¹H NMR (400 MHz, DMSO-d₆) δ 8.91 (t, J=6.1 Hz, 1H), 8.68 (s, 1H), 8.43(d, J=9.1 Hz, 2H), 8.31 (d, J=8.9 Hz, 1H), 8.01-8.10 (m, 3H), 7.77 (d,J=9.1 Hz, 2H), 7.60 (d, J=7.8 Hz, 1H), 7.39 (s, 1H), 7.33 (s, 1H), 6.99(s, 3H), 6.89 (s, 1H), 6.85 (d, J=9.1 Hz, 2H), 4.04 (dd, J=3.9, 8.9 Hz,1H), 3.98 (s, 3H), 3.92 (s, 3H), 3.41-3.45 (m, 2H), 3.27-3.38 (m, 6H),3.23 (m, 12H), 3.06 (s, 3H), 2.85-2.95 (m, 1H), 2.72-2.81 (m, 1H),2.52-2.56 (m, 2H), 2.17 (t, J=7.3 Hz, 2H), 1.74-1.88 (m, 4H). HR-ESI-MS:C₅₉H₆₂N₁₁O₁₁S^(|) [M+H]⁺, 1132.43455; found 1132.43384.

Example 2 Labeling of a Cysteine Derivative with Compounds of theInvention

General Procedure

To a vial containing 985 μL of PBS (1×, pH 7.6), were subsequently added5 μL of the stock solution of benzamide (10 mM in water), 5 μL of thestock solution of arylpropiolonitrile (1-12, 10 mM in DMSO) and 5 μL ofstock solution of AcCysNHBn (7 m, 10 mM in DMSO). Aliquots of thereaction mixture (50 μL) were analyzed by HPLC (injection at 0 and 30minutes of reaction). Areas under the peaks of the starting materialsand hydrolysis products were normalized according to the area of thepeak of the internal standard.

Results

Obtained results are summed up in table 2 below, which presents theconversion of the compounds 1-12 in 30 minutes in presence of 7m at 50μM concentration of each reagent and 25° C. The reaction is extremelysensitive to steric hindrances induced by substituents in ortho-positionto propiolonitrile group (entries 1, 5, 8-9, 4) as well as to electroniceffect of the substituent: −I and −M substituents increase (entries 10and 12), while +M substituents decrease the reactivity of the compound(entries 3 and 7).

TABLE 2 compound Conversion compound Conversion 1 o-OMe 10.1% 10 o-NO₂70.4%* 2 m-OMe 46.8% 12 p-CONHMe 85.5% 3 p-OMe 14.6% 11 p-NHAc 52.3% 5o-NH₂ 10.8% 8 o-Me 10.3% 6 m-NH₂ 42.0% 9 o,o′-diMe 2.3%** 7 p-NH₂ 7.4% 4o,o′-diOMe 4.8%** *Byproducts were observed; **conversion in 60 minutes.

Example 3 Hydrolytic Stability of a Compound of Formula (I) andComparison with Phenylmaleimide

To a vial containing 980 μL of PBS (1×, pH 7.6), were subsequently added10 μL of a stock solution of benzamide and 10 μL of a stock solution ofelectrophile (phenylmaleimide 1 or

11) to give final concentration of 1 mM (both internal standard andelectrophile). Aliquots of the reaction mixture (50 μL) were analyzed byHPLC for 5 hours of hydrolysis (injection every 30 min). Areas under thepeaks of the starting materials and hydrolysis products were normalizedaccording to the area of the peak of the internal standard.

The obtained results are presented in FIGS. 4 and 5. Noticeablehydrolysis was observed only for phenylmaleimide 1 (PhMal,k_(obs)=7×10⁻⁵ s⁻¹). 11 showed no detectable change in concentration.

Example 4 Stability of Compounds of Formula (III)

Stability of the Following Compound in Different Conditions

A 100 mM stock solution of the “addition product”

was prepared in DMSO and stored at −20° C. 1 μL of the stock solutionwas added to 999 μL of working solutions to give 100 μM finalconcentration of substrate. Aliquots were analyzed at 0, 30 and 60 min.Areas under peak of starting material were normalized according to thearea of the peak of the internal standard (benzamide). All measurementswere carried at 25° C.

Table 3 below shows the conversion of “the addition product” indifferent media in one hour.

TABLE 3 Conversion of “addition # Working solution product” in 1 h A 100mM PhSH in PBS(7.4):DMSO = 80:20 0.7% B 1M H2O2 1.8% C 1M HCl (pH = 0)0.1% D 1M NaOH (pH = 14) 3.4% E 1M Glutathione reduced (GSH) in PBS(7.4) 0.4% F 1M Imidazole in PBS (7.4) 0.5%

This experiment clearly show that the addition product is stable andundergoes very little degradation in a wide range of conditions, inparticular for pH ranging from 0 to 14.

In addition, hardly any thiol exchange is observed when the additionproduct is exposed for 1 hour to a medium comprising an excess ofanother thiol, such as phenylthiol or glutathione.

Stability of a Compound of Formula (III) and Comparison with Maleimide

The stability of compounds A (according to the invention) and B(reference compound) below was studied in different biologicalconditions.

Cell Culture:

Normal liver BNL CL.2 cells from mouse were grown in Dulbecco's MEMmedium with 1 g/l glucose (Eurobio, Les Ulis, France) supplemented with10% fetal bovine serum (Perbio, Brebieres, France), 2 mM L-Glutamine,100 U/mL penicillin, 100 μg/mL streptomycin (Eurobio). Cells weremaintained in a 5% CO₂ humidified atmosphere at 37° C.

Microscopy:

Twenty four hours prior to experiment, 2.5×10⁴ BNL CL.2 cells wereseeded per well in 8-well Lab-Tek II Chambered coverglass plates (ref155409, Nunc, Naperville, Ill., USA). The required amounts of probes Aand B were diluted up to 300 μl in MEM complete medium to give finalconcentration of 1 μM and then added onto the cells. A 5 μg/ml ofHoechst 118 solution was used as a nuclear marker. Cells were observedwith a confocal Leica TSC SPE II microscope after washing with 10% FBSred phenol free Eagle's MEM medium.

Cytometry:

The day before experiment, BNL CL.2 cells were seeded in 96-plates(Greiner Bio One, Frickenhausen, Germany) at 2.0×10⁴ cells/well inDulbecco's MEM complete medium. Both probes (A and B) were prepared at 1μM concentrations in Dulbecco's MEM complete and added onto cells duringdifferent times (2, 6 and 24 hours). After washing with PBS (Eurobio), 5min incubation with 40 μl trypsine, and addition of 160 μl of PBS EDTA 5mM, cells were analyzed by flow cytometry on a PCA-96 Guava cytometer(Guava Technologies Merck Millipore, Billerica, Mass., USA) with a greenlaser.

First, compound A was far more stable than compound B in human plasma(see FIG. 1). Second, compound A was far more stable than compound B incellulo (see FIG. 2).

Example 4 Selectivity of Compounds of the Invention Towards the ThiolMoiety

Screening for selectivity was done on benzylamides of non-protectedamino acids. 100 mM stock solutions of benzylamides of amino acids (inform of TFA salts) and electrophiles (phenylpropiolonitrile andphenylmaleimide) were prepared in DMSO and stored at −20° C. A 100 mMstock solution of benzamide (used as an internal standard) was preparedin distilled water and stocked at −20° C. Analyses of reaction mixtureswere conducted with Shimadzu LC with SunFire™ C18 5 μM 4.6×150 mm column(Waters). HPLC parameters were as follows: flow rate 1 mL/min, gradientfrom 5 to 95% of mobile phase B from 0 to 20 min, followed by 5 min at95% of mobile phase and post time of 5 min. Mobile phase A was 0.05% TFAin water (mQ) (v/v), and mobile phase B was acetonitrile (HPLC grade).Data were analyzed using Shimadzu analysis software. Signals werenormalized according to the area of the peak of the internal standard(benzamide). Areas under the peaks of the amino acid benzylamides wereused to calculate their conversion during reaction.

10 μL of the stock solution of amino acid benzamide and 2.5 μL of thestock solution of benzamide were added to a vial containing 977.5 μL ofPBS (1×, pH 7.6). The solution was stirred and 10 μL of the stocksolution of electrophile were added to give 1 mM final concentrations ofreagents and 0.25 mM concentration of benzamide. Aliquots of thereaction mixture (50 μL) were analyzed by HPLC for 1 hour of hydrolysis(injections at 0, 30 and 60 min). Areas under the peaks of the startingmaterials and hydrolysis products were normalized according to the areaof peak of the internal standard. In case of phenylpropiolonitrile, noneof amino acid models gave more than 1.6% conversion (see table 3 below).Conversely, when phenylmaleimide was tested, some amino acidbenzylamides showed conversions up to 8.5%. Masses of correspondingadducts were detected by mass spectrometry (ESI-LCMS) in some cases(shown in bold, Table 4).

Table 4 below presents the conversion of benzylamides in 1 hour in PBS(1×, pH 7.6) in presence of phenylpropiolonitrile (1).

TABLE 4 Amino acid benzamide Conversion CysNHBn (7a)  98% GlyNHBn (7b)0.2% AlaNHBn (7c) 0.1% ValNHBn (7d) 0.7% SerNHBn (7e) 0.3% MetNHBn (7f)0.1% TyrNHBn (7g) 1.6% HisNHBn (7h) 0.6% GlnNHBn (7i) 0.1% TrpNHBn (7j)1.3% ArgNHBn (7k) 0.4% AspNHBn (7l) 0.6%

Table 5 below presents the conversion of benzylamides in 1 hour in PBS(1×, pH 7.6) in presence of phenylmaleimide. Bold values correspond tothose for which the mass of the corresponding adduct was detected byESI-LCMS.

TABLE 5 Amino acid benzamide Conversion CysNHBn (7a) 100%  GlyNHBn (7b)3.3% AlaNHBn (7c) 0.2% ValNHBn (7d) 0.9% SerNHBn (7e) 2.8% MetNHBn (7f)1.9% TyrNHBn (7g) 2.4% HisNHBn (7h) 8.5% GlnNHBn (7i) 1.7% TrpNHBn (7j)1.0% ArgNHBn (7k) 2.9% AspNHBn (7l) 1.3%

In conclusion, the selectivity of compounds of the invention towards thethiol moiety when compared to other moieties is clearly higher than theselectivity obtained for corresponding compounds, wherein thepropiolonitrile moiety is replaced with a maleimide moiety.

Example 5 Toxicity Tests

Toxicity of the following “linker” compounds of formula (II) was studiedby MTT assay on HaCaT cell lines:

In vitro cytotoxicity was measured using an MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.The experiments were performed in 96-well plates with HaCaT cells grownto confluence in cell culture media (RPMI 1640 media supplemented with10% fetal calf serum and 1 mM Glutamin, 200 μL per well). Cells wereincubated with chemical reagent at different concentrations (100 μM to0.78 μM by serial ½ dilutions) at 37° C. for 24 hours. After incubation,the supernatant was replaced with fresh culture media containing MTT(300 μg/mL). After 2 hours of incubation at 37° C., the media wascarefully removed and 100 μL of DMSO were added to dissolve the formazancrystals generated by mitochondrial enzymes-induced reduction of theMTT. The absorbance was measured at 595 nm using a microplate reader(Biotek, Synergy HT). The cell viabilities were expressed as percent ofuntreated control cells.

The results of the MTT test are presented on FIG. 5, and clearly showthat compounds of formula (II) are not toxic and may thus be used forinstance for biological applications.

Example 6 Labeling of Lysozyme with a Compound of Formula (I)

Labeling of Tryptic Digest of Lysozyme

1 nmol of lysozyme was solubilized in NH₄HCO₃ (25 mM) and reduced with 1mM of TCEP at 57° C. during 1 hour. A solution of APN-TMPP (1 mM inDMSO) was added to the protein at a molar ratio of 1:200. Then, labeledprotein was subjected to proteolysis by porcine trypsin (Promega V5111).Sample was digested with 1:100 (w/w) trypsin in 25 mM ammoniumbicarbonate at 35° C. overnight. NanoLC-MS/MS analyses were performed tofollow the reaction. The resulting peptide mixtures were analyzed by C18reversed phase nanoHPLC on a nanoACQUITY Ultra-Performance-LC system(Waters, Milford, Mass.) coupled to a Q-TOF maXis (Bruker Daltonics,Bremen, Germany) mass spectrometer equipped with a nano-electrospraysource. Chromatographic separation was performed on a nanoACQUITYUltra-Performance-LC. The peptides were separated on an ACQUITY UPLC®BEH130 C18 column (Waters Corp.), 75 μm×200 mm, 1.7 μm particle size.The solvent system consisted of 0.1% formic acid in water (solvent A)and 0.1% formic acid in acetonitrile (solvent B). Trapping was performedon a 20×0.18 mm, 5 μm Symmetry C18 pre-column (Waters Corp.) during 3minutes at 5 μL/min with 99% of solvent A and 1% of solvent B. Elutionwas performed at a flow rate of 300 nL/min, using a 1-50% gradient ofsolvent B for 30 minutes at 50° C. followed by a fast rise at 80% (5minutes) of solvent B. The complete system was fully controlled byHystar 3.2 (Bruker Daltonics). The Q-TOF instrument was operated withthe following settings: source temperature was set to 200° C., dryinggaz flow was 4 l/h, and the nano-electrospray voltage was 4 kV. Masscalibration of the TOF was achieved using ES-TOF Tuning Mix (AgilentTechnologies) on the 50 to 2200 m/z range in positive mode. For tandemMS experiments, the system was operated with automatic switching betweenMS and MS/MS modes both on m/z range [50-2200]. In MS the summation timewas 0.2 s. In MS/MS summation time was weighted between 0.2 s and 1.4 sin function of parent ion intensity. The 2 most abundant peptides(intensity threshold 400 au), preferably ions with two, three, four orfive charges, were selected on each MS spectrum for further isolationand CID fragmentation with 2 energies set using collision energyprofile. Fragmentation was performed using argon as the collision gas.Tryptic peptides were manually sequenced (de novo) to confirm theirsequence and locate the cysteine tagged by the APN-TMPP probe. Thepeptides were identified using extracted ion chromatograms (EIC) basedon monoisotopic mass of calculated peptide sequences.

Evaluation of APN-TMPP chemoselectivity was carried by studying of itsreaction with tryptic digest at 200:1 molar ratio of APN-TMPP (1 μM) toprotein (around 10:1 to cysteine moieties) at room temperature for onehour. Peptide mixtures obtained without and with chemical derivatizationwere analyzed by LC-MS/MS. All detectable cysteine-containing peptidesreacted with a probe and were delayed while cysteine-free peptides wereunaffected. The labeling efficiency was evaluated based on the ratiobetween intensities of labeled and non-labeled peptides by LC-MS. Morethan 98% of the detected peptides were completely labeled. LC-MS resultsshow clearly that cysteine-containing peptides have an increasedretention time due to the addition of the hydrophobic TMPP group,whereas the retention time of all other peptides was unchanged (Table6).

Table 6 below shows the results of LC-MS analyses of tryptic digest oflysozyme before and after reaction with APN-TMPP.

TABLE 6 Before tagging After tagging m/z  RT m/z RT ΔRT NumberPeptide sequence^((a)) (charge state) (min) (charge state) (min) (min)of tags ²⁴ CELAAAMK³¹ 418.70 (+2) 13.9 545.89 (+3) 24.2 10.3 1 ²⁴CELAAAMoxK³¹ 426.70 (+2) 12.6 551.23 (+3) 23.7 11.1 1 ⁴⁰GYSLGNWVCAAK⁵¹634.81 (+2) 19.8 689.96 (+3) 25.3 5.5 1 ⁸⁰WWCNDGR⁸⁶ 468.69 (+2) 16.5579.22 (+3) 25.7 9.2 1 ⁹²NLCNIPCSALLASDITASVNCAK¹¹⁴ 779.71 (+2) 23.7949.20 (+5) 30.3 6.6 3 ⁵²FESNFNTQATNR⁶³ 714.83 (+2) 13.5 714.83 (+2)13.5 0 0 ¹³⁵GTDVQAWIR¹⁴³ 523.27 (+2) 17.3 523.27 (+7) 17.3 0 0⁶⁴NTDGSTDYGILQINSR⁷⁹ 585.28 (+2) 18.2 585.28 (+2) 18.2 0 0¹¹⁶IVSDGNGMNAWVAWR¹³⁰ 559.27 (+2) 20.9 559.27 (+2) 20.9 0 0^((a))Cysteine residues are in bold.

Example 7 Conjugation of Solubilizating APN Reagents (49-54) With CD38A275C Mutant

General scheme of the experiment (on the example of modification of CD38mutant with 49) is illustrated on FIG. 6.

To 300 μL of CD38C275 solution (1 mg/mL) was added 6 μL of 50 mMsolution of solubilizing APN reagent (49-54) in DMSO. In parallel, as acontrol, to 300 μL of CD38C275 was added 6 μL of DMSO. Both samples wereincubated for 15 hours at 25° C., then dialyzed 5 times (membrane cutoff of 10 k) to give final volume of 30 μL each (10 mg/mL). Size ofaggregates was measured by DLS.

Example 8 Labeling of CD38-CD375 Mutant and Comparison with Maleimide

a) Stability of the Compound of the Invention and of the CorrespondingMaleimide

The compounds below were synthesized:

Stability studies proved that the compound according to the inventionwas stable for 24 hours in PBS (Phosphate Buffer Saline). Comparatively,the corresponding compound comprising a maleimide moiety was 70%degraded after one hour in PBS.

b) Reaction with the CD38 Mutant

Both compounds were reacted with a 2 μM solution of the CD38-C375mutant.

Gel electrophoresis after purification showed that a higher labelingrate could be obtained with the compounds according to the inventionthan with the corresponding maleimide compound. FIG. 7 presents the gelelectrophoresis obtained with the compound of the invention and themaleimide, before and after purification.

Example 9 Conjugation of Trastuzumab and TAMRA Using Compound 18

General scheme of the experiment is illustrated on FIG. 8.

To the solution of Trastuzumab (100 uL, 10 mg/mL in 50 mM borate bufferpH 8.5) was added 1.74 uL of the solution of 18 (10 mg/mL in DMSO).After incubation for 1 h at 25° C. was added 0.69 uL of TAMRA-SH (100 mMin DMSO). The mixture was incubated at 25° C. for 16 h and the conjugatewas purified by size exclusion chromatography.

The comparison experiment was carried out using4-(N-maleimidomethyl)cyclohexanecarboxylic acid N-hydroxysuccinimideester (SMCC) instead of 18.

SDS-PAGE analysis of the obtained conjugates (FIG. 9) showed thatcompound 18 allows for higher levels of conjugation comparing to SMCC.

Native ESI-MS analysis of the conjugate prepared using 18 (FIG. 10, FIG.11) showed that in average one molecule of TAMRA per antibody wasconjugated.

Native ESI-MS analysis of the conjugate prepared using SMCC (FIG. 12,FIG. 13) showed a complex mixture of undistinguishable species.

Experiment shows that the compound 18 allows for higher levels ofconjugation and gives cleaner population of conjugates comparing togenerally applied SMCC.

Example 10 Direct Conjugation of the Compound 58 to Partially ReducedTrastuzumab

General scheme of the experiment is illustrated on FIG. 14.

To the solution of Trastuzumab (100 uL, 10 mg/mL in 50 mM PBS pH 7.4with 10 mM of EDTA) was added the solution of TCEP (10 mM in water, 1.1or 2.2 eq.). The mixture was incubated at 37° C. for 2 h and then thesolution of 58 (8.25 μL, 10 mM in DMSO) was added. The mixture wasincubated at 25° C. for 16 h and the conjugate was purified by sizeexclusion chromatography.

SDS-PAGE analysis of the obtained conjugates (FIG. 15) showed thatcompound 58 was covalently attached to the antibody. ESI-MS analysisshowed that in average 4 molecules were conjugated per antibody using2.2 eq. of TCEP.

Example 11 Rebridging of Antibody Fragments Using Compounds 33 and 34

General scheme of the experiment is illustrated on FIG. 16.

To the solution of Trastuzumab (100 uL, 10 mg/mL in 50 mM PBS pH 7.4with 10 mM of EDTA) was added the solution of TCEP (10 mM in water, 5eq.). The mixture was incubated at 37° C. for 2 h and then the solutionof 33 or 34 (10 mM in DMSO, 15 eq.) was added. The resulting solutionwas incubated for 16 h at 25° C. and then analyzed by SDS-PAGE inreducing conditions.

SDS-PAGE analysis showed that antibody fragments were successfullybridged by compounds 33 and 34 (FIG. 17).

The invention claimed is:
 1. A compound of formula (III):

wherein each of R₁, R₂, R₄, and R₅ is a hydrogen atom; R₃ is afluorescent probe or a drug, which is bounded to the phenyl ring via alinker; and R₆ is such that R₆—SH is a protein containing a thiolmoiety.